Novel type vi crispr orthologs and systems

ABSTRACT

The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered RNA-targeting systems comprising a novel RNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a U.S. national stage application based onInternational Application No. PCT/US2018/027125 filed Apr. 11, 2018,which claims priority to U.S. Provisional Application No. 62/484,791filed Apr. 12, 2017, U.S. Provisional Application No. 62/561,662 filedSep. 21, 2017, and U.S. Provisional Application No. 62/568,129 filedOct. 4, 2017, each of which is incorporated herein by reference in itsentirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbersMH100706 and MH110049 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

SEQUENCE LISTING

The substitute electronic sequence listing (“BROD-4770US_ST25.txt;” Sizeis 1,177,921 bytes and it was created on Aug. 31, 2020) is hereinincorporated by reference in its entirety and replaces any allpreviously submitted Sequence Listings.

Reference is made to PCT application including as it designates the US,inter alia, application No. PCT/US2016/058302, filed Oct. 21, 2016.Reference is made to U.S. provisional patent application 62/245,270filed on Oct. 22, 2015, U.S. provisional patent application 62/296,548filed on Feb. 17, 2016, and U.S. provisional patent applications62/376,367 and 62/376,382, filed on Aug. 17, 2016. Reference is furthermade to U.S. 62/471,792, filed Mar. 15, 2017. Reference is further madeto U.S. provisional patent application 62/471,170, filed Mar. 17, 2017.Reference is further made to U.S. provisional patent application62/484,791, filed Apr. 12, 2017. Reference is further made to U.S.provisional patent application 62/561,662, filed Sep. 21, 2017. Mentionis made of: Smargon et al. (2017), “Cas13b Is a Type VI-BCRISPR-Associated RNA-Guided RNase Differentially Regulated by AccessoryProteins Csx27 and Csx28,” Molecular Cell 65, 618-630 (Feb. 16, 2017)doi: 10.1016/j.molce1.2016.12.023. Epub Jan. 5, 2017 and Smargon et al.(2017), “Cas13b Is a Type VI-B CRISPR-Associated RNA-Guided RNaseDifferentially Regulated by Accessory Proteins Csx27 and Csx28,” bioRxiv092577; doi: https://doi.org/10.1101/092577. Posted Dec. 9, 2017. Eachof the foregoing applications and literature citations are herebyincorporated herein by reference.

All documents cited or referenced in herein cited documents, togetherwith any manufacturer's instructions, descriptions, productspecifications, and product sheets for any products mentioned herein orin any document incorporated by reference herein, are herebyincorporated herein by reference, and may be employed in the practice ofthe invention. More specifically, all referenced documents areincorporated by reference to the same extent as if each individualdocument was specifically and individually indicated to be incorporatedby reference.

FIELD OF THE INVENTION

The present invention generally relates to systems, methods andcompositions used for the control of gene expression involving sequencetargeting, such as perturbation of gene transcripts or nucleic acidediting, that may use vector systems related to Clustered RegularlyInterspaced Short Palindromic Repeats (CRISPR) and components thereof.

BACKGROUND OF THE INVENTION

Recent advances in genome sequencing techniques and analysis methodshave significantly accelerated the ability to catalog and map geneticfactors associated with a diverse range of biological functions anddiseases. Precise genome targeting technologies are needed to enablesystematic reverse engineering of causal genetic variations by allowingselective perturbation of individual genetic elements, as well as toadvance synthetic biology, biotechnological, and medical applications.Although genome-editing techniques such as designer zinc fingers,transcription activator-like effectors (TALEs), or homing meganucleasesare available for producing targeted genome perturbations, there remainsa need for new genome and transcriptome engineering technologies thatemploy novel strategies and molecular mechanisms and are affordable,easy to set up, scalable, and amenable to targeting multiple positionswithin the eukaryotic genome and transcriptome. This would provide amajor resource for new applications in genome engineering andbiotechnology.

The CRISPR-Cas systems of bacterial and archaeal adaptive immunity showextreme diversity of protein composition and genomic loci architecture.The CRISPR-Cas system loci has more than 50 gene families and there isno strictly universal genes indicating fast evolution and extremediversity of loci architecture. So far, adopting a multi-prongedapproach, there is comprehensive cas gene identification of about 395profiles for 93 Cas proteins. Classification includes signature geneprofiles plus signatures of locus architecture. A new classification ofCRISPR-Cas systems is proposed in which these systems are broadlydivided into two classes, Class 1 with multisubunit effector complexesand Class 2 with single-subunit effector modules exemplified by the Cas9protein. Novel effector proteins associated with Class 2 CRISPR-Cassystems may be developed as powerful genome engineering tools and theprediction of putative novel effector proteins and their engineering andoptimization is important.

The CRISPR-Cas adaptive immune system defends microbes against foreigngenetic elements via DNA or RNA-DNA interference. Class 2 type VIsingle-component CRISPR-Cas effectors target RNA. One such is Cas13a(also known as C2c2; see Shmakov et al. (2015) “Discovery and FunctionalCharacterization of Diverse Class 2 CRISPR-Cas Systems”; Molecular Cell60:1-13; doi: http://dx.doi.org/10.1016/j.molce1.2015.10.008), which wascharacterized as an RNA-guided Rnase (Abudayyeh et al. (2016), Science,[Epub ahead of print], June 2; “C2c2 is a single-component programmableRNA-guided RNA-targeting CRISPR effector”; doi:10.1126/science.aaf5573). Under current classification, Cas13a is aClass 2 type VI-A CRISPR-Cas system. An alternative is provided byCas13b, a Class 2 Type VI-B effector protein. Class 2 Type VI-B effectorproteins include two subgroups, Type VI-B1 and Type VI-B2, which arealso referred to as Group 29 proteins and Group 30 proteins, and includemembers which are RNA-programmable nucleases, RNA-interfering and may beinvolved in bacterial adoptive immunity against RNA phages. (see SmargonA et al. “Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNAsedifferentially regulated by accessory proteins Csx27 and Csx28”,Molecular Cell, online Jan. 5, 2017. DOI: 10.1016/j.molce1.2016.12.023).

Group 29 and group 30 systems comprise a large single effector(approximately 1100 amino acids in length), termed Cas13b, and one ornone of two small putative accessory proteins (approximately 200 aminoacids in length, and termed Csx27 and Csx28) nearby a CRISPR array.Based on the nearby small protein, the system is classified as Type VI-B1 (Csx27) or Type VI-B2 (Csx28). No additional proteins out to 25kilobase pairs upstream or downstream from the array are conservedacross species with each locus. With minor exceptions, the CRISPR arraycomprises direct repeat sequences 36 nucleotides in length and spacersequences 30 nucleotides in length. The direct repeat is generally wellconserved, especially at the ends, with a GTTG/GUUG at the 5′ endreverse complementary to a CAAC at the 3′ end. This conservationsuggests strong base pairing for an RNA loop structure that potentiallyinteracts with the protein(s) in the locus. A motif search complementaryto the direct repeats revealed no candidate tracrRNAs nearby the arrays,indicative of a single crRNA like that found in the Cpf1 locus.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

There exists a pressing need for alternative and robust systems andtechniques for targeting nucleic acids or polynucleotides (e.g. DNA orRNA or any hybrid or derivative thereof) with a wide array ofapplications, in particular in eukaryotic systems, more in particular inmammalian systems. This invention addresses this need and providesrelated advantages. Adding the novel RNA-targeting systems of thepresent application to the repertoire of genomic, transcriptomic, andepigenomic targeting technologies may transform the study andperturbation or editing of specific target sites through directdetection, analysis and manipulation, in particular in eukaryoticsystems, more in particular in mammalian systems (including cells,organs, tissues, or organisms). To utilize the RNA-targeting systems ofthe present application effectively for RNA targeting withoutdeleterious effects, it is critical to understand aspects of engineeringand optimization of these RNA targeting tools.

The Class 2 type VI-B effector protein Cas13b is a RNA-guided RNase thatcan be efficiently programmed to degrade ssRNA. The present inventorshave undertaken screening to identify a representative number of Cas13borthologs from different species, and to determine efficacy of thoseorthologs in eukaryotic cellular environments. In various embodiments,the invention refers to, includes, or makes use of a Type VI-BCRISPR-Cas effector protein, or a Cas13b effector protein; as well as tonucleic acids encoding such proteins.

In some embodiments, the effector protein is at least 50%, 60%, 70%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or morehomologous or identical to a wild type Cas13b effector protein of aprokaryotic organism selected from the group consisting ofPorphyromonas, Prevotella, Bacteroides, Riemerella, Bergeyella,Alistipes, Myroides, Capnocytophaga, and Flavobacterium. In someembodiments, the effector protein is at least 50%, 60%, 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous oridentical to a wild type Cas13b effector protein of a prokaryoticorganism selected from the group consisting of Porphyromonas gulae,Prevotella sp., Porphyromonas gingivalis, Bacteroides pyogenes,Riemerella anatipestifer, Bergeyella zoohelcum, Prevotella intermedia,Prevotella buccae, Alistipes sp., Prevotella aurantiaca, Myroidesodoratimimus, Capnocytophaga canimorsus, Flavobacterium branchiophilum,and Flavobacterium columnare. In preferred embodiments, the effectorprotein is at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more homologous or identical to a wild typeCas13b effector protein selected from the group consisting ofPorphyromonas gulae Cas13b (accession number WP_039434803), Prevotellasp. P5-125 Cas13b (accession number WP_044065294), Porphyromonasgingivalis Cas13b (accession number WP_053444417), Porphyromonas sp.COT-052 OH4946 Cas13b (accession number WP_039428968), Bacteroidespyogenes Cas13b (accession number WP_034542281), Riemerellaanatipestifer Cas13b (accession number WP_004919755). The most preferredeffector proteins are those at least 50%, 60%, 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous or identicalto a wild type Cas13b effector protein selected from the groupconsisting of Porphyromonas gulae Cas13b (accession numberWP_039434803), Prevotella sp. P5-125 Cas13b (accession numberWP_044065294), Porphyromonas gingivalis Cas13b (accession numberWP_053444417), Porphyromonas sp. COT-052 OH4946 Cas13b (accession numberWP_039428968); and most specifically preferred are Porphyromonas gulaeCas13b (accession number WP_039434803) or Prevotella sp. P5-125 Cas13b(accession number WP_044065294). The full amino acid sequences of eachof these Cas13b effector proteins, and others, is given in FIG. 1.

In some embodiments, the Cas13b effector protein (a) comprises 900-1800amino acids and two HEPN domains, (b) is naturally present in aprokaryotic genome within 10 kb upstream or downstream of a CRISPRarray, (c) is the only encoded protein comprising more than 700 aminoacids within 10 kb upstream or downstream of the CRISPR array, and/or(d) there is no Cas1 gene or Cas2 gene within 10 kb upstream ordownstream of the CRISPR array. In some embodiments, at least one ofCsx27 or Csx28 is also present within 10 kb upstream or downstream ofthe CRISPR array.

In certain embodiments, the Cas13b effector protein has a modifiedsequence when compared to a wild-type protein. In certain embodiments,the effector protein is identical to a wild type Cas13b effector proteinin at least one or more common motifs shared among two or more Cas13beffector proteins. Common motifs may be determined by standard sequencealignment tools to identify consensus sequences. In particularembodiments, the Cas13b effector protein is a protein comprising asequence having at least 70% sequence identity with one or more of thesequences consisting of DKHXFGAFLNLARHN (SEQ ID NO:1), GLLFFVSLFLDK (SEQID NO:2), SKIXGFK (SEQ ID NO:3), DMLNELXRCP (SEQ ID NO:4),RXZDRFPYFALRYXD (SEQ ID NO: 5) and LRFQVBLGXY (SEQ ID NO:6). In furtherparticular embodiments, the Cas13b effector protein comprises a sequencehaving at least 70% sequence identity at least 2, 3, 4, 5 or all 6 ofthese sequences. In further particular embodiments, the sequenceidentity with these sequences is at least 75%, 80%, 85%, 90%, 95% or100%. In further particular embodiments, the Cas 13b effector protein isa protein comprising a sequence having 100% sequence identity withGLLFFVSLFL (SEQ ID NO:7) and RHQXRFPYF (SEQ ID NO:8). In furtherparticular embodiments, the Cas13b effector is a Cas13b effector proteincomprising a sequence having 100% sequence identity with RHQDRFPY (SEQID NO:9).

It will be appreciated that the terms Cas enzyme, CRISPR enzyme, CRISPRprotein Cas protein and CRISPR Cas are generally used interchangeablyand at all points of reference herein refer by analogy to CRISPReffector proteins further described in this application, unlessotherwise apparent, such as by specific reference to Cas9.

In embodiments of the invention, a Type VI-B system comprises a Cas13beffector protein and optionally a small accessory protein encodedupstream or downstream of the Cas13b effector protein. In certainembodiments, the small accessory protein enhances the Cas13b effector'sability to target RNA.

In certain embodiments of the invention, a Type VI-B system comprises aCas13b effector protein and optionally a small accessory protein encodedupstream or downstream of the Cas13b effector protein. In certainembodiments, the small accessory protein represses the Cas13b effector'sability to target RNA.

The invention provides a non-naturally occurring or engineeredcomposition comprising i) a Type VI-B CRISPR-Cas effector protein, andii) a Type VI-B CRISPR-Cas crRNA, wherein the crRNA comprises a) a guidesequence that is capable of hybridizing to a target RNA sequence, and b)a direct repeat sequence. The Type VI-B CRISPR-Cas effector proteinforms a complex with the crRNA, and the guide sequence directssequence-specific binding of the complex to the target RNA sequence,whereby there is formed a CRISPR complex comprising the Type VI-BCRISPR-Cas effector protein complexed with the guide sequence that ishybridized to the target RNA sequence. The complex that is formed whenthe guide sequence hybridizes to the target RNA sequence includesinteraction (recognition) of the protospacer flanking sequence (PFS).

In some embodiments, a non-naturally occurring or engineered compositionof the invention may comprise a Type VI-B CRISPR-Cas accessory proteinthat enhances Type VI-B CRISPR-Cas effector protein activity. In certainsuch embodiments, the accessory protein that enhances Type VI-BCRISPR-Cas effector protein activity is a csx28 protein. In suchembodiments, the Type VI-B CRISPR-Cas effector protein and the Type VI-BCRISPR-Cas accessory protein may be from the same source or from adifferent source.

In some embodiments, a non-naturally occurring or engineered compositionof the invention comprises a Type VI-B CRISPR-Cas accessory protein thatrepresses Type VI-B CRISPR-Cas effector protein activity. In certainsuch embodiment, the accessory protein that represses Type VI-BCRISPR-Cas effector protein activity is a csx27 protein. In suchembodiments, the Type VI-B CRISPR-Cas effector protein and the Type VI-BCRISPR-Cas accessory protein may be from the same source or from adifferent source.

In some embodiments, a non-naturally occurring or engineered compositionof the invention comprises two or more Type VI-B CRISPR-Cas crRNAs.

In some embodiments, a non-naturally occurring or engineered compositionof the invention comprises a guide sequence that hybridizes to a targetRNA sequence in a prokaryotic cell. In some embodiments, a non-naturallyoccurring or engineered composition of the invention comprises a guidesequence that hybridizes to a target RNA sequence in a eukaryotic cell.The CRISPR system as provided herein can make use of a crRNA oranalogous polynucleotide comprising a guide sequence, wherein thepolynucleotide is an RNA, a DNA or a mixture of RNA and DNA, and/orwherein the polynucleotide comprises one or more nucleotide analogs. Thesequence can comprise any structure, including but not limited to astructure of a native crRNA, such as a bulge, a hairpin or a stem loopstructure. In certain embodiments, the polynucleotide comprising theguide sequence forms a duplex with a second polynucleotide sequencewhich can be an RNA or a DNA sequence.

In certain embodiments, the methods make use of chemically modifiedguide RNAs. Examples of guide RNA chemical modifications include,without limitation, incorporation of 2′-O-methyl (M), 2′-O-methyl3′phosphorothioate (MS), or 2′-O-methyl 3′thioPACE (MSP) at one or moreterminal nucleotides. Such chemically modified guide RNAs can compriseincreased stability and increased activity as compared to unmodifiedguide RNAs, though on-target vs. off-target specificity is notpredictable. (See, Hendel, 2015, Nat Biotechnol. 33(9):985-9, doi:10.1038/nbt.3290, published online 29 Jun. 2015). Chemically modifiedguide RNAs further include, without limitation, RNAs withphosphorothioate linkages and locked nucleic acid (LNA) nucleotidescomprising a methylene bridge between the 2′ and 4′ carbons of theribose ring.

In some embodiments, the Type VI-B CRISPR-Cas effector protein comprisesone or more nuclear localization signals (NLSs).

Cas13b achieves RNA cleavage through conserved basic residues within itstwo HEPN domains, in contrast to the catalytic mechanisms of other knownRNases found in CRISPR-Cas systems. Mutation of the HEPN domain, such as(e.g. alanine) substitution of any of the four predicted HEPN domaincatalytic residues can convert Cas13b into an inactive programmableRNA-binding protein (dCas13b, analogous to dCas9).

The ability of dCas13b to bind to specified sequences could be used inseveral aspects according to the invention to (i) bring effector modulesto specific transcripts to modulate the function or translation, whichcould be used for large-scale screening, construction of syntheticregulatory circuits and other purposes; (ii) fluorescently tag specificRNAs to visualize their trafficking and/or localization; (iii) alter RNAlocalization through domains with affinity for specific subcellularcompartments; and (iv) capture specific transcripts (through direct pulldown of dCas13b or use of dCas13b to localize biotin ligase activity tospecific transcripts) to enrich for proximal molecular partners,including RNAs and proteins.

Active Cas13b should also have many applications. An aspect of theinvention involves targeting a specific transcript for destruction. Inaddition, Cas13b, once primed by the cognate target, can cleave other(non-complementary) RNA molecules in vitro and can inhibit cell growthin vivo, Biologically, this promiscuous RNase activity may reflect aprogrammed cell death/dormancy (PCD/D)-based protection mechanism of thetype VI-B CRISPR-Cas systems. Accordingly, in an aspect of theinvention, it might be used to trigger PCD or dormancy in specificcells—for example, cancer cells expressing a particular transcript,neurons of a given class, cells infected by a specific pathogen, orother aberrant cells or cells the presence of which is otherwiseundesirable.

The invention provides a method of modifying nucleic acid sequencesassociated with or at a target locus of interest, in particular ineukaryotic cells, tissues, organs, or organisms, more in particular inmammalian cells, tissues, organs, or organisms, the method comprisingdelivering to said locus a non-naturally occurring or engineeredcomposition comprising a Type VI-B CRISPR-Cas loci effector protein andone or more nucleic acid components, wherein the effector protein formsa complex with the one or more nucleic acid components and upon bindingof the said complex to the locus of interest the effector proteininduces the modification of the sequences associated with or at thetarget locus of interest. In a preferred embodiment, the modification isthe introduction of a strand break. In a preferred embodiment, thesequences associated with or at the target locus of interest compriseRNA and the effector protein is encoded by a type VI-B CRISPR-Cas locus.The complex can be formed in vitro or ex vivo and introduced into a cellor contacted with RNA; or can be formed in vivo.

The invention provides a method of targeting (such as modifying)sequences associated with or at a target locus of interest, the methodcomprising delivering to said sequences associated with or at the locusa non-naturally occurring or engineered composition comprising a Cas13bloci effector protein (which may be catalytically active, oralternatively catalytically inactive) and one or more nucleic acidcomponents, wherein the Cas13b effector protein forms a complex with theone or more nucleic acid components and upon binding of the said complexto the locus of interest the effector protein induces the modificationof sequences associated with or at the target locus of interest. In apreferred embodiment, the modification is the introduction of a strandbreak. In a preferred embodiment the Cas13b effector protein forms acomplex with one nucleic acid component; advantageously an engineered ornon-naturally occurring nucleic acid component. The complex can beformed in vitro or ex vivo and introduced into a cell or contacted withRNA; or can be formed in vivo. The induction of modification ofsequences associated with or at the target locus of interest can beCas13b effector protein-nucleic acid guided. In a preferred embodimentthe one nucleic acid component is a CRISPR RNA (crRNA). In a preferredembodiment the one nucleic acid component is a mature crRNA or guideRNA, wherein the mature crRNA or guide RNA comprises a spacer sequence(or guide sequence) and a direct repeat sequence or derivatives thereof.In a preferred embodiment the spacer sequence or the derivative thereofcomprises a seed sequence, wherein the seed sequence is critical forrecognition and/or hybridization to the sequence at the target locus.

Aspects of the invention relate to Cas13b effector protein complexeshaving one or more non-naturally occurring or engineered or modified oroptimized nucleic acid components. In a preferred embodiment the nucleicacid component of the complex may comprise a guide sequence linked to adirect repeat sequence, wherein the direct repeat sequence comprises oneor more stem loops or optimized secondary structures. In one embodiment,the direct repeat sequence may be about 36 nucleotides in length. In aspecific embodiment, the direct repeat comprises a GTTG/GUUG at the 5′end that is reverse complementary to a CAAC at the 3′ end. In certainembodiments, the direct repeat has a minimum length of 16 nts, such asat least 28 nt, and a single stem loop. In further embodiments thedirect repeat has a length longer than 16 nts, preferably more than 17nts, such as at least 28 nt, and has more than one stem loop oroptimized secondary structures. In particular embodiments, the directrepeat has 25 or more nts, such as 26 nt, 27 nt, 28 nt or more, and oneor more stem loop structures. In a preferred embodiment the directrepeat may be modified to comprise one or more protein-binding RNAaptamers. In a preferred embodiment, one or more aptamers may beincluded such as part of optimized secondary structure. Such aptamersmay be capable of binding a bacteriophage coat protein. Thebacteriophage coat protein may be selected from the group comprising Qβ,F2, GA, fr, JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1,TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb 12r, ϕCb23r,7s and PRR1. In a preferred embodiment the bacteriophage coat protein isMS2. The invention also provides for the nucleic acid component of thecomplex being 30 or more, 40 or more or 50 or more nucleotides inlength.

The invention provides cells comprising Cas13b effector protein and/orguides and or complexes thereof with target nucleic acids, includingcells comprising transiently expressed or introduced Cas13b effectorprotein and/or guides and or complexes thereof. In certain embodiments,the cell is a eukaryotic cell, including but not limited to a yeastcell, a plant cell, a mammalian cell, an animal cell, or a human cell.

The invention also provides a method of modifying a target locus ofinterest, in particular in eukaryotic cells, tissues, organs, ororganisms, more in particular in mammalian cells, tissues, organs, ororganisms, the method comprising delivering to said locus anon-naturally occurring or engineered composition comprising a Cas13bloci effector protein and one or more nucleic acid components, whereinthe Cas13b effector protein forms a complex with the one or more nucleicacid components and upon binding of the said complex to the locus ofinterest the effector protein induces the modification of the targetlocus of interest. In a preferred embodiment, the modification is theintroduction of a strand break. The complex can be formed in vitro or exvivo and introduced into a cell or contacted with RNA; or can be formedin vivo.

In such methods the target locus of interest may be comprised within anRNA molecule. Also, the target locus of interest may be comprised withina DNA molecule, and in certain embodiments, within a transcribed DNAmolecule. In such methods the target locus of interest may be comprisedin a nucleic acid molecule in vitro.

In such methods the target locus of interest may be comprised in anucleic acid molecule within a cell, in particular a eukaryotic cell,such as a mammalian cell or a plant cell. The mammalian cell many be anon-human primate, bovine, porcine, rodent or mouse cell. The cell maybe a non-mammalian eukaryotic cell such as poultry, fish or shrimp. Theplant cell may be of a crop plant such as cassava, corn, sorghum, wheat,or rice. The plant cell may also be of an algae, tree or vegetable. Themodification introduced to the cell by the present invention may be suchthat the cell and progeny of the cell are altered for improvedproduction of biologic products such as an antibody, starch, alcohol orother desired cellular output. The modification introduced to the cellby the present invention may be such that the cell and progeny of thecell include an alteration that changes the biologic product produced.

The mammalian cell may be a non-human mammal, e.g., primate, bovine,ovine, porcine, canine, rodent, Leporidae such as monkey, cow, sheep,pig, dog, rabbit, rat or mouse cell. The cell may be a non-mammalianeukaryotic cell such as poultry bird (e.g., chicken), vertebrate fish(e.g., salmon) or shellfish (e.g., oyster, clam, lobster, shrimp) cell.The cell may also be a plant cell. The plant cell may be of a monocot ordicot or of a crop or grain plant such as cassava, corn, sorghum,soybean, wheat, oat or rice. The plant cell may also be of an algae,tree or production plant, fruit or vegetable (e.g., trees such as citrustrees, e.g., orange, grapefruit or lemon trees; peach or nectarinetrees; apple or pear trees; nut trees such as almond or walnut orpistachio trees; nightshade plants; plants of the genus Brassica; plantsof the genus Lactuca; plants of the genus Spinacia; plants of the genusCapsicum; cotton, tobacco, asparagus, carrot, cabbage, broccoli,cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry,blueberry, raspberry, blackberry, grape, coffee, cocoa, etc).

The invention provides a method of modifying a target locus of interest,the method comprising delivering to said locus a non-naturally occurringor engineered composition comprising a Type VI-B CRISPR-Cas locieffector protein and one or more nucleic acid components, wherein theeffector protein forms a complex with the one or more nucleic acidcomponents and upon binding of the said complex to the locus of interestthe effector protein induces the modification of the target locus ofinterest. In a preferred embodiment, the modification is theintroduction of a strand break.

The invention also provides a method of modifying a target locus ofinterest, the method comprising delivering to said locus a non-naturallyoccurring or engineered composition comprising a Cas13b loci effectorprotein and one or more nucleic acid components, wherein the Cas13beffector protein forms a complex with the one or more nucleic acidcomponents and upon binding of the said complex to the locus of interestthe effector protein induces the modification of the target locus ofinterest. In a preferred embodiment, the modification is theintroduction of a strand break.

In such methods the target locus of interest may be comprised in anucleic acid molecule in vitro. In such methods the target locus ofinterest may be comprised in a nucleic acid molecule within a cell.Preferably, in such methods the target locus of interest may becomprised in a RNA molecule in vitro. Also preferably, in such methodsthe target locus of interest may be comprised in a RNA molecule within acell. The cell may be a prokaryotic cell or a eukaryotic cell. The cellmay be a mammalian cell. The cell may be a rodent cell. The cell may bea mouse cell.

In any of the described methods the target locus of interest may be agenomic or epigenomic locus of interest. In any of the described methodsthe complex may be delivered with multiple guides for multiplexed use.In any of the described methods more than one protein(s) may be used.

In further aspects of the invention the nucleic acid components maycomprise a CRISPR RNA (crRNA) sequence. Without limitation, theApplicants hypothesize that in such instances, the pre-crRNA maycomprise secondary structure that is sufficient for processing to yieldthe mature crRNA as well as crRNA loading onto the effector protein. Bymeans of example and not limitation, such secondary structure maycomprise, consist essentially of or consist of a stem loop within thepre-crRNA, more particularly within the direct repeat.

In any of the described methods the effector protein and nucleic acidcomponents may be provided via one or more polynucleotide moleculesencoding the protein and/or nucleic acid component(s), and wherein theone or more polynucleotide molecules are operably configured to expressthe protein and/or the nucleic acid component(s). The one or morepolynucleotide molecules may comprise one or more regulatory elementsoperably configured to express the protein and/or the nucleic acidcomponent(s). The one or more polynucleotide molecules may be comprisedwithin one or more vectors. In any of the described methods the targetlocus of interest may be a genomic or epigenomic locus of interest. Inany of the described methods the complex may be delivered with multipleguides for multiplexed use. In any of the described methods more thanone protein(s) may be used.

Regulatory elements may comprise inducible promotors. Polynucleotidesand/or vector systems may comprise inducible systems.

In any of the described methods the one or more polynucleotide moleculesmay be comprised in a delivery system, or the one or more vectors may becomprised in a delivery system.

In any of the described methods the non-naturally occurring orengineered composition may be delivered via liposomes, particlesincluding nanoparticles, exosomes, microvesicles, a gene-gun or one ormore viral vectors.

The invention also provides a non-naturally occurring or engineeredcomposition which is a composition having the characteristics asdiscussed herein or defined in any of the herein described methods.

In certain embodiments, the invention thus provides a non-naturallyoccurring or engineered composition, such as particularly a compositioncapable of or configured to modify a target locus of interest, saidcomposition comprising a Type VI-B CRISPR-Cas loci effector protein andone or more nucleic acid components, wherein the effector protein formsa complex with the one or more nucleic acid components and upon bindingof the said complex to the locus of interest the effector proteininduces the modification of the target locus of interest. In certainembodiments, the effector protein may be a Cas13b loci effector protein.

The invention also provides in a further aspect a non-naturallyoccurring or engineered composition, such as particularly a compositioncapable of or configured to modify a target locus of interest, saidcomposition comprising: (a) a guide RNA molecule (or a combination ofguide RNA molecules, e.g., a first guide RNA molecule and a second guideRNA molecule, such as for multiplexing) or a nucleic acid encoding theguide RNA molecule (or one or more nucleic acids encoding thecombination of guide RNA molecules); (b) a Type VI-B CRISPR-Cas locieffector protein or a nucleic acid encoding the Type VI-B CRISPR-Casloci effector protein. In certain embodiments, the effector protein maybe a Cas13b loci effector protein.

The invention also provides in a further aspect a non-naturallyoccurring or engineered composition comprising: (a) a guide RNA molecule(or a combination of guide RNA molecules, e.g., a first guide RNAmolecule and a second guide RNA molecule) or a nucleic acid encoding theguide RNA molecule (or one or more nucleic acids encoding thecombination of guide RNA molecules); (b) be a Cas13b loci effectorprotein.

The invention also provides a vector system comprising one or morevectors, the one or more vectors comprising one or more polynucleotidemolecules encoding components of a non-naturally occurring or engineeredcomposition which is a composition having the characteristics as definedin any of the herein described methods.

The invention also provides a delivery system comprising one or morevectors or one or more polynucleotide molecules, the one or more vectorsor polynucleotide molecules comprising one or more polynucleotidemolecules encoding components of a non-naturally occurring or engineeredcomposition which is a composition having the characteristics discussedherein or as defined in any of the herein described methods.

The invention also provides a non-naturally occurring or engineeredcomposition, or one or more polynucleotides encoding components of saidcomposition, or vector or delivery systems comprising one or morepolynucleotides encoding components of said composition for use in atherapeutic method of treatment. The therapeutic method of treatment maycomprise gene or transcriptome editing, or gene therapy.

The invention also provides for methods and compositions wherein one ormore amino acid residues of the effector protein may be modified e.g.,an engineered or non-naturally-occurring effector protein or Cas13b. Inan embodiment, the modification may comprise mutation of one or moreamino acid residues of the effector protein. The one or more mutationsmay be in one or more catalytically active domains of the effectorprotein. The effector protein may have reduced or abolished nucleaseactivity compared with an effector protein lacking said one or moremutations. The effector protein may not direct cleavage of the RNAstrand at the target locus of interest. In a preferred embodiment, theone or more mutations may comprise two mutations. In a preferredembodiment the one or more amino acid residues are modified in a Cas13beffector protein, e.g., an engineered or non-naturally-occurringeffector protein or Cas13b. In certain embodiments, the effector proteincomprises one or more of the following mutations: R116A, H121A, R1177A,H1182A (wherein amino acid positions correspond to amino acid positionsof Cas13b protein originating from Bergeyella zoohelcum ATCC 43767),such as R116A, H121A, R1177A, and H1182A; R116A, H121A, and R1177A;R116A, H121A, and H1182A; R116A, R1177A, and H1182A; H121A, R1177A, andH1182A; R116A and H121A; R116A and R1177A; R116A and H1182A; H121A andR1177A; H121A and H1182A; R1177A and H1182A; R116A; H121A; R1177A;H1182A. The skilled person will understand that corresponding amino acidpositions in different Cas13b proteins may be mutated to the sameeffect. In certain embodiments, one or more of mutations R116A, H121A,R1177A, H1182A abolish catalytic activity of the protein completely orpartially (e.g. altered cleavage rate, altered specificity, etc.), suchas R116A, H121A, R1177A, and H1182A; R116A, H121A, and R1177A; R116A,H121A, and H1182A; R116A, R1177A, and H1182A; H121A, R1177A, and H1182A;R116A and H121A; R116A and R1177A; R116A and H1182A; H121A and R1177A;H121A and H1182A; R1177A and H1182A; R116A; H121A; R1177A; H1182A. Incertain embodiments, wherein amino acid positions correspond to aminoacid positions of Cas13b protein originating from Prevotella sp. P5-125,the effector protein comprises H133A and H1058A mutations. In certainembodiments, the effector protein as described herein is a “dead”effector protein, such as a dead Cas13b effector protein (i.e. dCas13b).In certain embodiments, the effector protein has one or more mutationsin HEPN domain 1. In certain embodiments, the effector protein has oneor more mutations in HEPN domain 2. In certain embodiments, the effectorprotein has one or more mutations in HEPN domain 1 and HEPN domain 2.The effector protein may comprise one or more heterologous functionaldomains. The one or more heterologous functional domains may compriseone or more nuclear localization signal (NLS) domains. The one or moreheterologous functional domains may comprise at least two or more NLSdomains. The one or more NLS domain(s) may be positioned at or near orin proximity to a terminus of the effector protein (e.g., Cas13beffector protein) and if two or more NLSs, each of the two may bepositioned at or near or in proximity to a terminus of the effectorprotein (e.g., Cas13b effector protein). The one or more heterologousfunctional domains may comprise one or more transcriptional activationdomains. In a preferred embodiment the transcriptional activation domainmay comprise VP64. The one or more heterologous functional domains maycomprise one or more transcriptional repression domains. In a preferredembodiment the transcriptional repression domain comprises a KRAB domainor a SID domain (e.g. SID4×). The one or more heterologous functionaldomains may comprise one or more nuclease domains. In a preferredembodiment a nuclease domain comprises Fok1.

The invention also provides for the one or more heterologous functionaldomains to have one or more of the following activities: methylaseactivity, demethylase activity, translation activation activity,translation repression activity, transcription activation activity,transcription repression activity, transcription release factoractivity, histone modification activity, nuclease activity,single-strand RNA cleavage activity, double-strand RNA cleavageactivity, single-strand DNA cleavage activity, double-strand DNAcleavage activity and nucleic acid binding activity. At least one ormore heterologous functional domains may be at or near theamino-terminus of the effector protein and/or wherein at least one ormore heterologous functional domains is at or near the carboxy-terminusof the effector protein. The one or more heterologous functional domainsmay be fused to the effector protein. The one or more heterologousfunctional domains may be tethered to the effector protein. The one ormore heterologous functional domains may be linked to the effectorprotein by a linker moiety.

In certain embodiments of the invention, the one or more heterologousfunctional domains may comprise epitope tags or reporters. Non-limitingexamples of epitope tags include histidine (His) tags, V5 tags, FLAGtags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, andthioredoxin (Trx) tags. Examples of reporters include, but are notlimited to, glutathione-S-transferase (GST), horseradish peroxidase(HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase,beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed,DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP),and autofluorescent proteins including blue fluorescent protein (BFP).

The invention also provides for the effector protein comprising aneffector protein which is at least 50%, 60%, 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous or identicalto a wild type Cas13b effector protein of a prokaryotic genus selectedfrom the group consisting of Porphyromonas, Prevotella, Bacteroides,Riemerella, Bergeyella, Alistipes, Myroides, Capnocytophaga, andFlavobacterium. The invention further provides for the effector proteincomprising an effector protein which is at least 50%, 60%, 70%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologousor identical to a wild type Cas13b effector protein of a prokaryoticspecies selected from the group consisting of Porphyromonas gulae,Prevotella sp., Porphyromonas gingivalis, Bacteroides pyogenes,Riemerella anatipestifer, Bergeyella zoohelcum, Prevotella intermedia,Prevotella buccae, Alistipes sp., Prevotella aurantiaca, Myroidesodoratimimus, Capnocytophaga canimorsus, Flavobacterium branchiophilum,and Flavobacterium columnare. The invention additionally provides forthe effector protein comprising an effector protein which is at least50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more homologous or identical to a wild type Cas13b effectorprotein selected from the group consisting of Porphyromonas gulae Cas13b(accession number WP_039434803), Prevotella sp. P5-125 Cas13b (accessionnumber WP_044065294), Porphyromonas gingivalis Cas13b (accession numberWP_053444417), Porphyromonas sp. COT-052 OH4946 Cas13b (accession numberWP_039428968), Bacteroides pyogenes Cas13b (accession numberWP_034542281), Riemerella anatipestifer Cas13b (accession numberWP_004919755). The most preferred effector proteins are those at least50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more homologous or identical to a wild type Cas13b effectorprotein selected from the group consisting of Porphyromonas gulae Cas13b(accession number WP_039434803), Prevotella sp. P5-125 Cas13b (accessionnumber WP_044065294), Porphyromonas gingivalis Cas13b (accession numberWP_053444417), Porphyromonas sp. COT-052 OH4946 Cas13b (accession numberWP_039428968); and most specifically preferred are those at least 50%,60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore homologous or identical to a wild type Porphyromonas gulae Cas13b(accession number WP_039434803) or Prevotella sp. P5-125 Cas13b(accession number WP_044065294). The effector protein may comprise achimeric effector protein comprising a first fragment from a firsteffector protein ortholog and a second fragment from a second effectorprotein ortholog, and wherein the first and second effector proteinorthologs are different.

In certain embodiments, the effector protein may be at least 700 aminoacids long. In preferred embodiments, the effector protein may be about900 to about 1500 amino acids long, e.g., about 900 to about 1000 aminoacids long, about 1000 to about 1100 amino acids long, about 1100 toabout 1200 amino acids long, or about 1200 to about 1300 amino acidslong, or about 1300 to about 1400 amino acids long, or about 1400 toabout 1500 amino acids long, e.g., about 900, about 1000, about 1100,about 1200, about 1300, about 1400, about 1500, about 1600, about 1700,or about 1800 amino acids long.

In some embodiments, the Cas13b effector protein (a) comprises 900-1800amino acids and two HEPN domains, (b) is naturally present in aprokaryotic genome within 10 kb upstream or downstream of a CRISPRarray, (c) is the only protein comprising more than 700 amino acidswithin 10 kb upstream or downstream of the CRISPR array, and/or (d)there is no Cas1 gene or Cas2 gene within 10 kb upstream or downstreamof the CRISPR array. In some embodiments, Csx27 or Csx28 is also presentwithin 10 kb upstream or downstream of the CRISPR array.

In certain embodiments, the effector protein, particularly a Type VI-Bloci effector protein, more particularly a Cas13b, comprises at leastone and preferably at least two, such as more preferably exactly two,conserved RxxxxH motifs. Catalytic RxxxxH motifs are are characteristicof HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding) domains.Hence, in certain embodiments, the effector proteincomprises at leastone and preferably at least two, such as more preferably exactly two,HEPN domains. In certain embodiments, the HEPN domains may possess RNAseactivity. In other embodiments, the HEPN domains may possess DNAseactivity.

In certain embodiments, the Cas13b effector proteins as intended hereinmay be associated with a locus comprising short CRISPR repeats between30 and 40 bp long, more typically between 34 and 38 bp long, even moretypically between 36 and 37 bp long, e.g., 30, 31, 32, 33, 34, 35, 36,37, 38, 39, or 40 bp long. In certain embodiments the CRISPR repeats arelong or dual repeats between 80 and 350 bp long such as between 80 and200 bp long, even more typically between 86 and 88 bp long, e.g., 80,81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 bp long

In certain embodiments, a protospacer adjacent motif (PAM) or PAM-likemotif directs binding of the effector protein (e.g. a Cas13b effectorprotein) complex as disclosed herein to the target locus of interest.The PAM may be referred to as a PFS, or protospacer flanking site. Insome embodiments, the PAM may be a 5′ PAM (i.e., located upstream of the5′ end of the protospacer). In other embodiments, the PAM may be a 3′PAM (i.e., located downstream of the 5′ end of the protospacer). Inother embodiments, both a 5′ PAM and a 3′ PAM are required. In certainembodiments of the invention, a PAM or PAM-like motif may not berequired for directing binding of the effector protein (e.g. a Cas13beffector protein). In certain embodiments, a 5′ PAM is D (i.e. A, G, orU). In certain embodiments, a 5′ PAM is D for Type VI-B1 effectors. SeeExample 1, Table 2. Methods exist to determine consensus 5′ and 3′ PAMsfor a given Cas13b system. In certain embodiments of the invention,cleavage at repeat sequences may generate crRNAs (e.g. short or longcrRNAs) containing a full spacer sequence flanked by a short nucleotide(e.g. 5, 6, 7, 8, 9, or 10 nt or longer if it is a dual repeat) repeatsequence at the 5′ end (this may be referred to as a crRNA “tag”) andthe rest of the repeat at the 3′end. In certain embodiments, targetingby the effector proteins described herein may require the lack ofhomology between the crRNA tag and the target 5′ flanking sequence. Thisrequirement may be similar to that described further in Samai et al.“Co-transcriptional DNA and RNA Cleavage during Type III CRISPR-CasImmunity” Cell 161, 1164-1174, May 21, 2015, where the requirement isthought to distinguish between bona fide targets on invading nucleicacids from the CRISPR array itself, and where the presence of repeatsequences will lead to full homology with the crRNA tag and preventautoimmunity.

In certain embodiments, the Cas13b effector protein is engineered andcan comprise one or more mutations that reduce or eliminate nucleaseactivity, thereby reducing or eliminating RNA interfering activity.Mutations can also be made at neighboring residues, e.g., at amino acidsnear those that participate in the nuclease activity. In someembodiments, one or more putative catalytic nuclease domains areinactivated and the effector protein complex lacks cleavage activity andfunctions as an RNA binding complex. In a preferred embodiment, theresulting RNA binding complex may be linked with one or more functionaldomains as described herein.

In certain embodiments, the effector protein (CRISPR enzyme; Cas13;effector protein) according to the invention as described herein is acatalytically inactive or dead Cas13 effector protein (dCas13). In someembodiments, the dCas13 effector comprises mutations in the nucleasedomain. In some embodiments, the dCas13 effector protein has beentruncated. In some embodiments, to reduce the size of a fusion proteinof the Cas13b effector and the one or more functional domains, theC-terminus of the Cas13b effector can be truncated while stillmaintaining its RNA binding function. For example, at least 20 aminoacids, at least 50 amino acids, at least 80 amino acids, or at least 100amino acids, or at least 150 amino acids, or at least 200 amino acids,or at least 250 amino acids, or at least 300 amino acids, or at least350 amino acids, or up to 120 amino acids, or up to 140 amino acids, orup to 160 amino acids, or up to 180 amino acids, or up to 200 aminoacids, or up to 250 amino acids, or up to 300 amino acids, or up to 350amino acids, or up to 400 amino acids, may be truncated at theC-terminus of the Cas13b effector. Specific examples of Cas13btruncations include C-terminal 4984-1090, C-terminal 41026-1090, andC-terminal 41053-1090, C-terminal 4934-1090, C-terminal 4884-1090,C-terminal 4834-1090, C-terminal 4784-1090, and C-terminal 4734-1090,wherein amino acid positions correspond to amino acid positions ofPrevotella sp. P5-125 Cas13b protein. See FIG. 15B.

In certain embodiments, the one or more functional domains arecontrollable, i.e. inducible.

In certain embodiments of the invention, the guide RNA or mature crRNAcomprises, consists essentially of, or consists of a direct repeatsequence and a guide sequence or spacer sequence. In certainembodiments, the guide RNA or mature crRNA comprises, consistsessentially of, or consists of a direct repeat sequence linked to aguide sequence or spacer sequence. In preferred embodiments of theinvention, the mature crRNA comprises a stem loop or an optimized stemloop structure or an optimized secondary structure. In preferredembodiments the mature crRNA comprises a stem loop or an optimized stemloop structure in the direct repeat sequence, wherein the stem loop oroptimized stem loop structure is important for cleavage activity. Incertain embodiments, the mature crRNA preferably comprises a single stemloop. In certain embodiments, the direct repeat sequence preferablycomprises a single stem loop. In certain embodiments, the cleavageactivity of the effector protein complex is modified by introducingmutations that affect the stem loop RNA duplex structure. In preferredembodiments, mutations which maintain the RNA duplex of the stem loopmay be introduced, whereby the cleavage activity of the effector proteincomplex is maintained. In other preferred embodiments, mutations whichdisrupt the RNA duplex structure of the stem loop may be introduced,whereby the cleavage activity of the effector protein complex iscompletely abolished.

The invention also provides for the nucleotide sequence encoding theeffector protein being codon optimized for expression in a eukaryote oreukaryotic cell in any of the herein described methods or compositions.In an embodiment of the invention, the codon optimized nucleotidesequence encoding the effector protein encodes any Cas13b discussedherein and is codon optimized for operability in a eukaryotic cell ororganism, e.g., such cell or organism as elsewhere herein mentioned, forinstance, without limitation, a yeast cell, or a mammalian cell ororganism, including a mouse cell, a rat cell, and a human cell ornon-human eukaryote organism, e.g., plant.

In certain embodiments of the invention, at least one nuclearlocalization signal (NLS) is attached to the nucleic acid sequencesencoding the Cas13b effector proteins. In preferred embodiments at leastone or more C-terminal or N-terminal NLSs are attached (and hencenucleic acid molecule(s) coding for the Cas13b effector protein caninclude coding for NLS(s) so that the expressed product has the NLS(s)attached or connected). In certain embodiments of the invention, atleast one nuclear export signal (NES) is attached to the nucleic acidsequences encoding the Cas13b effector proteins. In preferredembodiments at least one or more C-terminal or N-terminal NESs areattached (and hence nucleic acid molecule(s) coding for the Cas13beffector protein can include coding for NES(s) so that the expressedproduct has the NES(s) attached or connected). In a preferred embodimenta C-terminal and/or N-terminal NLS or NES is attached for optimalexpression and nuclear targeting in eukaryotic cells, preferably humancells. In a preferred embodiment, the codon optimized effector proteinis Cas13b and the spacer length of the guide RNA is from 15 to 35 nt. Incertain embodiments, the spacer length of the guide RNA is at least 16nucleotides, such as at least 17 nucleotides, preferably at least 18 nt,such as preferably at least 19 nt, at least 20 nt, at least 21 nt, or atleast 22 nt. In certain embodiments, the spacer length is from 15 to 17nt, from 17 to 20 nt, from 20 to 24 nt, eg. 20, 21, 22, 23, or 24 nt,from 23 to 25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt, from 27-30nt, from 30-35 nt, or 35 nt or longer. In certain embodiments of theinvention, the codon optimized effector protein is Cas13b and the directrepeat length of the guide RNA is at least 16 nucleotides. In certainembodiments, the codon optimized effector protein is Cas13b and thedirect repeat length of the guide RNA is from 16 to 20 nt, e.g., 16, 17,18, 19, or 20 nucleotides. In certain preferred embodiments, the directrepeat length of the guide RNA is 19 nucleotides.

The invention also encompasses methods for delivering multiple nucleicacid components, wherein each nucleic acid component is specific for adifferent target locus of interest thereby modifying multiple targetloci of interest. The nucleic acid component of the complex may compriseone or more protein-binding RNA aptamers. The one or more aptamers maybe capable of binding a bacteriophage coat protein. The bacteriophagecoat protein may be selected from the group comprising Qβ, F2, GA, fr,JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP,FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1. Ina preferred embodiment the bacteriophage coat protein is MS2. Theinvention also provides for the nucleic acid component of the complexbeing 30 or more, 40 or more or 50 or more nucleotides in length.

In a further aspect, the invention provides a eukaryotic cell comprisinga modified target locus of interest, wherein the target locus ofinterest has been modified according to in any of the herein describedmethods. A further aspect provides a cell line of said cell. Anotheraspect provides a multicellular organism comprising one or more saidcells.

In certain embodiments, the modification of the target locus of interestmay result in: the eukaryotic cell comprising altered expression of atleast one gene product; the eukaryotic cell comprising alteredexpression of at least one gene product, wherein the expression of theat least one gene product is increased; the eukaryotic cell comprisingaltered expression of at least one gene product, wherein the expressionof the at least one gene product is decreased; or the eukaryotic cellcomprising an edited genome.

In certain embodiments, the eukaryotic cell may be a mammalian cell or ahuman cell.

In further embodiments, the non-naturally occurring or engineeredcompositions, the vector systems, or the delivery systems as describedin the present specification may be used for: site-specific geneknockout; site-specific genome editing; RNA sequence-specificinterference; or multiplexed genome engineering.

Also provided is a gene product from the cell, the cell line, or theorganism as described herein. In certain embodiments, the amount of geneproduct expressed may be greater than or less than the amount of geneproduct from a cell that does not have altered expression or editedgenome. In certain embodiments, the gene product may be altered incomparison with the gene product from a cell that does not have alteredexpression or edited genome.

Also provided is an engineered and non-naturally occurring eukaryoticcell, comprising at least one of (i) a Cas13b effector protein asdescribed herein, or (ii) a guide RNA capable of forming a CRISPR-Cascomplex with a Cas13b effector protein. In some embodiments, (i) and/or(ii) are transiently expressed or introduced into the cell. Alsoprovided are organisms comprising such cells, cell lines, progeny ofsaid cell lines or organisms. The organism may be a vertebrate, forexample a mammal. Alternatively, the organism may be a plant or afungus.

In a further aspect, the invention provides a eukaryotic cell comprisinga nucleotide sequence encoding the CRISPR system described herein whichensures the generation of a modified target locus of interest, whereinthe target locus of interest is modified according to in any of theherein described methods. A further aspect provides a cell line of saidcell. Another aspect provides a multicellular organism comprising one ormore said cells.

In certain embodiments, the modification of the target locus of interestmay result in: the eukaryotic cell comprising altered (protein)expression of at least one gene product; the eukaryotic cell comprisingaltered (protein) expression of at least one gene product, wherein the(protein) expression of the at least one gene product is increased; theeukaryotic cell comprising altered (protein) expression of at least onegene product, wherein the (protein) expression of the at least one geneproduct is decreased; or the eukaryotic cell comprising an editedtranscriptome.

In certain embodiments, the eukaryotic cell may be a mammalian cell or ahuman cell.

In further embodiments, the non-naturally occurring or engineeredcompositions, the vector systems, or the delivery systems as describedin the present specification may be used for RNA sequence-specificinterference, RNA sequence specific modulation of expression (inludingisoform specific expression), stability, localization, functionality(e.g. ribosomal RNAs or miRNAs), etc.; or multiplexing of suchprocesses.

In further embodiments, the non-naturally occurring or engineeredcompositions, the vector systems, or the delivery systems as describedin the present specification may be used for RNA detection and/orquantification in a sample, such as a biological sample. In certainembodiments, RNA detection is in a cell. In an embodiment, the inventionprovides a method of detecting a target RNA in a sample, comprising (a)incubating the sample with i) a Type VI-B CRISPR-Cas effector proteincapable of cleaving RNA, ii) a guide RNA capable of hybridizing to thetarget RNA, and iii) an RNA-based cleavage inducible reporter capable ofbeing non-specifically and detectably cleaved by the effector protein,(b) detecting said target RNA based on the signal generated by cleavageof said RNA-based cleavage inducible reporter.

In an embodiment the Type VI-B CRISPR-Cas effector protein is a Cas13beffector protein; for example, a Cas13b effector protein as describedherein. In an embodiment, the RNA-based cleavage inducible reporterconstruct comprises a fluorochrome and a quencher. In certainembodiments, the sample comprises a cell-free biological sample. Inother embodiments, the sample comprises or a cellular sample, forexample, without limitation a plant cell, or an animal cell. In anembodiment of the invention, the target RNA comprises a pathogen RNA,including, but not limited to a target RNA from a virus, bacteria,fungus, or parasite. In an embodiment, the guide RNA is designed todetect a target RNA which comprises a single nucleotide polymorphism ora splice variant of an RNA transcript. In an embodiment, the guide RNAcomprises one or more mismatched nucleotides with the target RNA. Incertain embodiments, the guide RNA hybridizes to aa target molecule thatis diagnostic for a disease state, such as, but not limited to, cancer,or an immune disease.

The invention provides a ribonucleic acid (RNA) detection system,comprising a) a Type VI-B CRISPR-Cas effector protein capable ofcleaving RNA, b) a guide RNA capable of binding to a target RNA, and c)an RNA-based cleavage inducible reporter capable of beingnon-specifically and detectably cleaved by the effector protein.Further, the invention provides a kit for RNA detection, which comprisesa) a Type VI-B CRISPR-Cas effector protein capable of cleaving RNA, andb) an RNA-based cleavage inducible reporter capable of beingnon-specifically and detectably cleaved by the effector protein. Incertain embodiments, the RNA-based cleavage inducible reporter constructcomprises a fluorochrome and a quencher.

In further embodiments, the non-naturally occurring or engineeredcompositions, the vector systems, or the delivery systems as describedin the present specification may be used for generating disease modelsand/or screening systems.

In further embodiments, the non-naturally occurring or engineeredcompositions, the vector systems, or the delivery systems as describedin the present specification may be used for: site-specifictranscriptome editing or purturbation; nucleic acid sequence-specificinterference; or multiplexed genome engineering.

In aspects of the invention, the Cas13b effector proteins, or systemsdescribed herein, may be used in the treatment, prevention, prophylaxis,or suppression of viral pathogenesis, infection, or propagation in amammalian subject. Aspects of the invention provide a Cas13b CRISPRsystem comprising (a) a Cas13b CRISPR effector protein and/or apolynucleic acid encoding a Cas13b CRISPR effector protein and (b) oneor more guide RNAs and/or one or more polynucleic acids encoding one ormore guide RNAs designed to bind to one or more target molecules of avirus for use in treating, preventing, suppressing, and/or alleviatingviral pathogenesis, infection and/or propagation in a subject. TheCas13b effector protein may be as herein defined, including as topreferred wild-type Cas13b proteins and preferred derivatives andmodifications thereof.

In some embodiments, the Cas13b effector proteins, or systems describedherein, may be used in the treatment, prevention, prophylaxis, orsuppression of Lassa virus pathogenesis, infection, or propagation in amammalian subject. Lassa virus is associated with DCs and vascularendothelial cells (see Kunz, S. et. al. 2005. Journal of Virology).

In some embodiments, the Cas13b effector proteins, or systems describedherein, may be used in the treatment, prevention, prophylaxis, orsuppression of Ebola virus pathogenesis, infection, or propagation in amammalian subject. Ebola virus is associated with numerous tissues andcell types including DCs, macrophages, hepatocytes, etc. (see Martines,R. B. et. al. 2015. Journal of Pathology).

In some embodiments, the Cas13b effector proteins, or systems describedherein, may be used in the treatment, prevention, prophylaxis, orsuppression of SARS-CoV pathogenesis, infection, or propagation in amammalian subject. SARS-CoV is associated with lung tissues and cells(see To, K F. et. al. 2004. Journal of Pathology).

In some embodiments, the Cas13b effector proteins, or systems describedherein, may be used in the treatment, prevention, prophylaxis, orsuppression of Zika virus pathogenesis, infection, or propagation in amammalian subject. Zika virus is associated with numerous tissues andcell types, including bodily fluids, placenta, brain, etc. (see Miner,J. J. & Diamond, M. S. 2017. Cell Host & Microbe).

In some embodiments, the Cas13b effector proteins, or systems describedherein, may be used in the treatment, prevention, prophylaxis, orsuppression of Dengue virus pathogenesis, infection, or propagation in amammalian subject. Dengue virus is associated with numerous tissues andcell types, including DCs, macrophages, liver, etc. (see Flipse, J. et.al. 2016. Journal of General Virology).

In some embodiments, the Cas13b effector proteins, or systems describedherein, may be used in the treatment, prevention, prophylaxis, orsuppression of Chikungunya virus pathogenesis, infection, or propagationin a mammalian subject. Chikungunya virus is associated with numeroustissues and cell types, including immune cells, liver, central nervoussystem, etc. (see Schwartz, O. & Albert, M. L. 2010. Nature Reviews).

In some embodiments, the Cas13b effector proteins, or systems describedherein, may be used in the treatment, prevention, prophylaxis, orsuppression of Influenza virus pathogenesis, infection, or propagationin a mammalian subject. Influenza virus is associated with lungepithelial cells and macrophages (see Medina, R. A. & Garcia-Sastre A.2011 Nature Reviews).

In some embodiments, the Cas13b effector proteins, or systems describedherein, may be used in the treatment, prevention, prophylaxis, orsuppression of HIV virus pathogenesis, infection, or propagation in amammalian subject. HIV virus is associated with T cells and macrophages(see Weiss, R. A. 2002. IUBMB Life).

In some embodiments, the Cas13b effector proteins, or systems describedherein, may be used in the treatment, prevention, prophylaxis, orsuppression of Rotavirus virus pathogenesis, infection, or propagationin a mammalian subject. Rotavirus virus is associated with intestinetissues and cells (see Lopez, S & Arias, C. F. 2006. CTMI).

In some embodiments, the Cas13b effector proteins, or systems describedherein, may be used in the treatment, prevention, prophylaxis, orsuppression of Herpes Simplex (HSV-1) pathogenesis, infection, orpropagation in a mammalian subject. HSV-1 is associated with epithelialcells and neuronal cells (see Schelhaas, M. et. al. 2003. Journal ofGeneral Virology).

In some embodiments, the Cas13b effector proteins, or systems describedherein, may be used in the treatment, prevention, prophylaxis, orsuppression of HCV pathogenesis, infection, or propagation in amammalian subject. HCV is associated with liver tissues and cells (seeDing, Q, et. al. 2014. Cell Host & Microbe).

In some embodiments, the Cas13b effector proteins, or systems describedherein, may be used in the treatment, prevention, prophylaxis, orsuppression of HBV pathogenesis, infection, or propagation in amammalian subject. HBV is associated with liver tissues and cells (seeSchieck, A. et. al. 2013. Hepatology).

Also provided is a gene product from the cell, the cell line, or theorganism as described herein. In certain embodiments, the amount of geneproduct expressed may be greater than or less than the amount of geneproduct from a cell that does not have altered expression or editedgenome. In certain embodiments, the gene product may be altered incomparison with the gene product from a cell that does not have alteredexpression or edited genome.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

Accordingly, it is an object of the invention not to encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. § 112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product. It may be advantageous in thepractice of the invention to be in compliance with Art. 53(c) EPC andRule 28(b) and (c) EPC. Nothing herein is to be construed as a promise.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 shows a list of wild-type Cas13b orthologs, as well as theiramino acid sequences, together with the source organism and the proteinaccession number.

FIG. 2 shows a classification of each of the Cas13b orthologs of FIG. 1with their relative efficacy in knockdown of luciferase expression inmammalian cell culture.

FIG. 3 compares activity of each of the active Cas13b orthologs,controlling for guide sequence.

FIG. 4 compares activity of two of the Cas13b orthologs—Porphyromonasgulae WP_039434803 and Prevotella sp. P5-125 WP_044065294—with activityof C2c2/Cas13a across various guide sequences.

FIG. 5: Characterization of a highly active Cas13b ortholog for RNAknockdown. (A) Schematic of stereotypical Cas13 loci and correspondingcrRNA structure. (B) Evaluation of 19 Cas13a, 15 Cas13b, and 7 Cas13corthologs for luciferase knockdown using two different guides. Orthologswith efficient knockdown using both guides are labeled with their hostorganism name. (C) PspCas13b and LwaCas13a knockdown activity arecompared by tiling guides against Gluc and measuring luciferaseexpression. (D) PspCas13b and LwaCas13a knockdown activity are comparedby tiling guides against Cluc and measuring luciferase expression. (E)Expression levels in log 2 (transcripts per million (TPM)) values of allgenes detected in RNA-seq libraries of non-targeting control (x-axis)compared to Gluc-targeting condition (y-axis) for LwaCas13a (red) andshRNA (black). Shown is the mean of three biological replicates. TheGluc transcript data point is labeled. (F) Expression levels in log 2(transcripts per million (TPM)) values of all genes detected in RNA-seqlibraries of non-targeting control (x-axis) compared to Gluc-targetingcondition (y-axis) for PspCas13b (blue) and shRNA (black). Shown is themean of three biological replicates. The Gluc transcript data point islabeled. (G) Number of significant off-targets from Gluc knockdown forLwaCas13a, PspCas13b, and shRNA from the transcriptome wide analysis inE and F.

FIG. 6: Engineering dCas13b-ADAR fusions for RNA editing. (A) Schematicof RNA editing by dCas13b-ADAR fusion proteins. (B) Schematic ofCypridina luciferase W85X target and targeting guide design. (C)Quantification of luciferase activity restoration for Cas13b-dADAR1(left) and Cas13b-ADAR2-cd (right) with tiling guides of length 30, 50,70, or 84 nt. (D) Schematic of target site for targeting Cypridinialuciferase W85X. (E) Sequencing quantification of A->I editing for 50 ntguides targeting Cypridinia luciferase W85X.

FIG. 7: Measuring sequence flexibility for RNA editing by REPAIRv1. (A)Schematic of screen for determining Protospacer Flanking Site (PFS)preferences of RNA editing by REPAIRv1. (B) Distributions of RNA editingefficiencies for all 4-N PFS combinations at two different editing sites(C) Quantification of the percent editing of REPAIRv1 at Cluc W85 acrossall possible 3 base motifs. (D) Heatmap of 5′ and 3′ base preferences ofRNA editing at Cluc W85 for all possible 3 base motifs

FIG. 8: Correction of disease-relevant mutations with REPAIRv1. (A)Schematic of target and guide design for targeting AVPR2 878G>A. (B) The878G>A mutation in AVPR2 is corrected to varying percentages usingREPAIRv1 with three different guide designs. (C) Schematic of target andguide design for targeting FANCC 1517G>A. (D) The 1517G>A mutation inFANCC is corrected to varying percentages using REPAIRv1 with threedifferent guide designs. (E) Quantification of the percent editing of 34different disease-relevant G>A mutations using REPAIRv1. (F) Analysis ofall the possible G>A mutations that could be corrected as annotated bythe ClinVar database. (G) The distribution of editing motifs for all G>Amutations in ClinVar is shown versus the editing efficiency by REPAIRv1per motif as quantified on the Gluc transcript.

FIG. 9: Characterizing specificity of REPAIRv1. (A) Schematic of KRAStarget site and guide design. (B) Quantification of percent editing fortiled KRAS-targeting guides. Editing percentages are shown at theon-target and neighboring adenosine sites. For each guide, the region ofduplex RNA is indicated by a red rectangle. (C) Transcriptome-wide sitesof significant RNA editing by REPAIRv1 with Cluc targeting guide. Theon-target site Cluc site (254 A>G) is highlighted in orange. (D)Transcriptome-wide sites of significant RNA editing by REPAIRv1 withnon-targeting guide.

FIG. 10: Rational mutagenesis of ADAR2 to improve the specificity ofREPAIRv1. (A) Quantification of luciferase signal restoration by variousdCas13-ADAR2 mutants as well as their specificity score plotted along aschematic for the contacts between key ADAR2 deaminase residues and thedsRNA target. The specificity score is defined as the ratio of theluciferase signal between targeting guide and non-targeting guideconditions. (B) Quantification of luciferase signal restoration byvarious dCas13-ADAR2 mutants versus their specificity score. (C)Measurement of the on-target editing fraction as well as the number ofsignificant off-targets for each dCas13-ADAR2 mutant by transcriptomewide sequencing of mRNAs. (D) Transcriptome-wide sites of significantRNA editing by REPAIRv1 and REPAIRv2 with a guide targeting apretermination site in Cluc. The on-target Cluc site (254 A>G) ishighlighted in orange. (E) RNA sequencing reads surrounding theon-target Cluc editing site (254 A>G) highlighting the differences inoff-target editing between REPAIRv1 and REPAIRv2. All A>G edits arehighlighted in red while sequencing errors are highlighted in blue. (F)RNA editing by REPAIRv1 and REPAIRv2 with guides targeting anout-of-frame UAG site in the endogenous KRAS and PPIB transcripts. Theon-target editing fraction is shown as a sideways bar chart on the rightfor each condition row. The duplex region formed by the guide RNA isshown by a red outline box.

FIG. 11: Bacterial screening of Cas13b orthologs for in vivo efficiencyand PFS determination. (A) Schematic of bacterial assay for determiningthe PFS of Cas13b orthologs. Cas13b orthologs with beta-lactamasetargeting spacers are co-transformed with beta-lactamase expressionplasmids and subjected to double selection. (B) Quantitation ofinterference activity of Cas13b orthologs targeting beta-lactamase asmeasured by colony forming units (cfu). (C) PFS logos for Cas13borthologs as determined by depleted sequences from the bacterial assay.

FIG. 12: Optimization of Cas13b knockdown and further characterizationof mismatch specificity. (A) Gluc knockdown with two different guides ismeasured using the top 2 Cas13a and top 4 Cas13b orthologs fused to avariety of nuclear localization and nuclear export tags. (B) Knockdownof KRAS is measured for LwaCas13a, RanCas13b, PguCas13b, and PspCas13bwith four different guides and compared to four position-matched shRNAcontrols. (C) Schematic of the single and double mismatch plasmidlibraries used for evaluating the specificity of LwaCas13a and PspCas13bknockdown. Every possible single and double mismatch is present in thetarget sequence as well as in 3 positions directly flanking the 5′ and3′ ends of the target site. (D) The depletion level of transcripts withthe indicated single mismatches are plotted as a heatmap for both theLwaCas13a and PspCas13b conditions. (E) The depletion level oftranscripts with the indicated double mismatches are plotted as aheatmap for both the LwaCas13a and PspCas13b conditions.

FIG. 13: Characterization of design parameters for dCas13-ADAR2 RNAediting. (A) Knockdown efficiency of Gluc targeting for wildtype Cas13band catalytically inactive H133A/H1058A Cas13b (dCas13b). (B)Quantification of luciferase activity restoration by dCas13b fused toeither the wildtype ADAR2 catalytic domain or the hyperactive E488Qmutant ADAR2 catalytic catalytic domain, tested with tiling Cluctargeting guides. (C) Guide design and sequencing quantification of A->Iediting for 30 nt guides targeting Cypridinia luciferase W85X. (D) Guidedesign and sequencing quantification of A->I editing for 50 nt guidestargeting PPIB. (E) Influence of linker choice on luciferase activityrestoration by REPAIRv1. (F) Influence of base identify opposite thetargeted adenosine on luciferase activity restoration by REPAIRv1.

FIG. 14: ClinVar motif distribution for G>A mutations. The number ofeach possible triplet motif observed in the ClinVar database for all G>Amutations.

FIG. 15: (A) Truncations of dCas13b still have functional RNA editing.Various N-terminal and C-terminal truncations of dCas13b allow for RNAediting as measured by restoration of luciferase signal. FIG. 15: (B)Further examples of dCas13b-ADAR constructs with different C-terminaltruncations of dCas13b.

FIG. 16: Comparison of other programmable ADAR systems with thedCas13-ADAR2 editor. (A) Schematic of two programmable ADAR schemes:BoxB-based targeting and full length ADAR2 targeting. In the BoxB scheme(top), the ADAR2 deaminase domain (ADAR2_(DD)(E488Q)) is fused to asmall bacterial virus protein called lambda N (λN), which bindsspecifically a small RNA sequence called BoxB-λ. A guide RNA containingtwo BoxB-λ, hairpins can then guide the ADAR2 D_(DD)(E488Q), −λN forsite specific editing. In the full length ADAR2 scheme (bottom), thedsRNA binding domains of ADAR2 bind a hairpin in the guide RNA, allowingfor programmable ADAR2 editing. (B) Transcriptome-wide sites ofsignificant RNA editing by BoxB-ADAR2 D_(DD)(E488Q) with a guidetargeting Cluc and a non-targeting guide. The on-target Cluc site (254A>G) is highlighted in orange. (C) Transcriptome-wide sites ofsignificant RNA editing by ADAR2 with a guide targeting Cluc and anon-targeting guide. The on-target Cluc site (254 A>G) is highlighted inorange. (D) Transcriptome-wide sites of significant RNA editing byREPAIRv1 with a guide targeting Cluc and a non-targeting guide. Theon-target Cluc site (254 A>G) is highlighted in orange. (E) Quantitationof on-target editing rate percentage for BoxB-ADAR2 D_(DD)(E488Q),ADAR2, and REPAIRv1 for targeting guides against Cluc. (F) Overlap ofoff-target sites between different targeting and non-targetingconditions for programmable ADAR systems.

FIG. 17: Efficiency and specificity of dCas13b-ADAR2 mutants. (A)Quantitation of luciferase activity restoration bydCas13b-ADAR2_(DD)(E488Q) mutants for Cluc-targeting and non-targetingguides. (B) Relationship between the ratio of targeting andnon-targeting guides and the number of RNA-editing off-targets asquantified by transcriptome-wide sequencing. (C) Quantification ofnumber of transcriptome-wide off-target RNA editing sites versuson-target Cluc editing efficiency for dCas13b-ADAR2_(DD)(E488Q) mutants.

FIG. 18: Transcriptome-wide specificity of RNA editing bydCas13b-ADAR2_(DD)(E488Q) mutants. (A) Transcriptome-wide sites ofsignificant RNA editing by dCas13b-ADAR2 D_(DD)(E488Q) mutants with aguide targeting Cluc. The on-target Cluc site (254 A>G) is highlightedin orange. (B) Transcriptome-wide sites of significant RNA editing bydCas13b-ADAR2 D_(DD)(E488Q) mutants with a non-targeting guide.

FIG. 19: Characterization of motif biases in the off-targets ofdCas13b-ADAR2_(DD)(E488Q) editing. (A) For eachdCas13b-ADAR2_(DD)(E488Q) mutant, the motif present across all A>Goff-target edits in the transcriptome is shown. (B) The distribution ofoff-target A>G edits per motif identity is shown for REPAIRv1 withtargeting and non-targeting guide. (C) The distribution of off-targetA>G edits per motif identity is shown for REPAIRv2 with targeting andnon-targeting guide.

FIG. 20: Further characterization of REPAIRv1 and REPAIRv2 off-targets.(A) Histogram of the number of off-targets per transcript for REPAIRv1.(B) Histogram of the number of off-targets per transcript for REPAIRv2.(C) Variant effect prediction of REPAIRv1 off targets. (D) Distributionof potential oncogenic effects of REPAIRv1 off targets. (E) Varianteffect prediction of REPAIRv2 off targets. (F) Distribution of potentialoncogenic effects of REPAIRv2 off targets.

FIG. 21: RNA editing efficiency and specificity of REPAIRv1 andREPAIRv2. (A) Quantification of percent editing of KRAS withKRAS-targeting guide 1 at the targeted adenosine and neighboring sitesfor REPAIRv1 and REPAIRv2. (B) Quantification of percent editing of KRASwith KRAS-targeting guide 3 at the targeted adenosine and neighboringsites for REPAIRv1 and REPAIRv2. (C) Quantification of percent editingof PPIB with PPIB-targeting guide 2 at the targeted adenosine andneighboring sites for REPAIRv1 and REPAIRv2.

FIG. 22: Demonstration of all potential codon changes with a A>G RNAeditor. (A) Table of all potential codon transitions enabled by A>Iediting. (B) A codon table demonstrating all the potential codontransitions enabled by A>I editing.

The figures herein are for illustrative purposes only and are notnecessarily drawn to scale.

DETAILED DESCRIPTION OF THE INVENTION

In general, a CRISPR-Cas or CRISPR system as used in the foregoingdocuments, such as WO 2014/093622 (PCT/US2013/074667) referscollectively to transcripts and other elements involved in theexpression of or directing the activity of CRISPR-associated (“Cas”)genes, including sequences encoding a Cas gene, a tracr(trans-activating CRISPR) sequence (e.g. tracrRNA or an active partialtracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and atracrRNA-processed partial direct repeat in the context of an endogenousCRISPR system), a guide sequence (also referred to as a “spacer” in thecontext of an endogenous CRISPR system), or “RNA(s)” as that term isherein used (e.g., RNA(s) to guide Cas, such as Cas9, e.g. CRISPR RNAand transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimericRNA)) or other sequences and transcripts from a CRISPR locus. Ingeneral, a CRISPR system is characterized by elements that promote theformation of a CRISPR complex at the site of a target sequence (alsoreferred to as a protospacer in the context of an endogenous CRISPRsystem).

When the CRISPR protein is a Class 2 Type VI-B effector (for example, aCas13b effector protein), a tracrRNA is not required. In an engineeredsystem of the invention, the direct repeat may encompassnaturally-occuring sequences or non-naturally-occurring sequences. Thedirect repeat of the invention is not limited to naturally occurringlengths and sequences. A direct repeat can be 36nt in length, but alonger or shorter direct repeat can vary. For example, a direct repeatcan be 30nt or longer, such as 30-100 nt or longer. For example, adirect repeat can be 30nt, 40nt, 50nt, 60nt, 70nt, 80nt, 90nt, 100nt, orlonger in length. In some embodiments, a direct repeat of the inventioncan include synthetic nucleotide sequences inserted between the 5′ and3′ ends of naturally occurring direct repeat. In certain embodiments,the inserted sequence may be self-complementary, for example, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or 100% self complementary. Furthermore, adirect repeat of the invention may include insertions of nucleotidessuch as an aptamer or sequences that bind to an adapter protein (forassociation with functional domains). In certain embodiments, one end ofa direct repeat containing such an insertion is roughly the first halfof a short DR and the other end is roughly the second half of the shortDR.

In the context of formation of a CRISPR complex, “target sequence”refers to a sequence to which a guide sequence is designed to havecomplementarity, where hybridization between a target sequence and aguide sequence promotes the formation of a CRISPR complex. A targetsequence may comprise RNA polynucleotides. In some embodiments, a targetsequence is located in the nucleus or cytoplasm of a cell. In someembodiments, direct repeats may be identified in silico by searching forrepetitive motifs that fulfill any or all of the following criteria: 1.found in a 2 Kb window of genomic sequence flanking the CRISPR locus; 2.span from 20 to 50 bp; and 3. interspaced by 20 to 50 bp. In someembodiments, 2 of these criteria may be used, for instance 1 and 2, 2and 3, or 1 and 3. In some embodiments, all 3 criteria may be used.

In embodiments of the invention the terms guide sequence and guide RNA,i.e. RNA capable of guiding Cas13b to a target genomic locus, are usedinterchangeably as in foregoing cited documents such as WO 2014/093622(PCT/US2013/074667). In general, a guide sequence is any polynucleotidesequence having sufficient complementarity with a target polynucleotidesequence to hybridize with the target sequence and directsequence-specific binding of a CRISPR complex to the target sequence. Insome embodiments, the degree of complementarity between a guide sequenceand its corresponding target sequence, when optimally aligned using asuitable alignment algorithm, is about or more than about 50%, 60%, 75%,80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may bedetermined with the use of any suitable algorithm for aligningsequences, non-limiting example of which include the Smith-Watermanalgorithm, the Needleman-Wunsch algorithm, algorithms based on theBurrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW,Clustal X, BLAT, Novoalign (Novocraft Technologies; available atwww.novocraft.com), ELAND (Illumina, San Diego, Calif.), SOAP (availableat soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). Insome embodiments, a guide sequence is about or more than about 5, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In someembodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30,25, 20, 15, 12, or fewer nucleotides in length. Preferably the guidesequence is 10-40 nucleotides long, such as 20-30 or 20-40 nucleotideslong or longer, such as 30 nucleotides long or about 30 nucleotideslong. In certain embodiments, the guide sequence is 10-30 nucleotideslong, such as 20-30 or 20-40 nucleotides long or longer, such as 30nucleotides long or about 30 nucleotides long for Cas13b effectors. Incertain embodiments, the guide sequence is 10-30 nucleotides long, suchas 20-30 nucleotides long, such as 30 nucleotides long or about 30nucleotides long for Cas13b effectors originating from Bergeyellazoohelcum (such as Bergeyella zoohelcum ATCC 43767). The ability of aguide sequence to direct sequence-specific binding of a CRISPR complexto a target sequence may be assessed by any suitable assay. For example,the components of a CRISPR system sufficient to form a CRISPR complex,including the guide sequence to be tested, may be provided to a hostcell having the corresponding target sequence, such as by transfectionwith vectors encoding the components of the CRISPR sequence, followed byan assessment of preferential cleavage within the target sequence, suchas by Surveyor assay as described herein. Similarly, cleavage of atarget polynucleotide sequence may be evaluated in a test tube byproviding the target sequence, components of a CRISPR complex, includingthe guide sequence to be tested and a control guide sequence differentfrom the test guide sequence, and comparing binding or rate of cleavageat the target sequence between the test and control guide sequencereactions. Other assays are possible, and will occur to those skilled inthe art.

In a classic CRISPR-Cas system, the degree of complementarity between aguide sequence and its corresponding target sequence can be about ormore than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%;a guide or RNA or sgRNA can be about or more than about 5, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,35, 40, 45, 50, 75, or more nucleotides in length; or guide or RNA orsgRNA can be less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, orfewer nucleotides in length. However, in certain aspects of theinvention, off-target interactions may be reduced, e.g., reduce theguide interacting with a target sequence having low complementarity.Indeed, certain mutations may result in the CRISPR-Cas system being ableto distinguish between target and off-target sequences that have greaterthan 80% to about 95% complementarity, e.g., 83%-84% or 88-89% or 94-95%complementarity (for instance, distinguishing between a target having 18nucleotides from an off-target of 18 nucleotides having 1, 2 or 3mismatches). Accordingly, in the context of the present invention thedegree of complementarity between a guide sequence and its correspondingtarget sequence may be greater than 94.5% or 95% or 95.5% or 96% or96.5% or 97% or 97.5% or 98% or 98.5% or 99% or 99.5% or 99.9%, or 100%.Off target is less than 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or98% or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% or 94% or93% or 92% or 91% or 90% or 89% or 88% or 87% or 86% or 85% or 84% or83% or 82% or 81% or 80% complementarity between the sequence and theguide, with it advantageous that off target is 100% or 99.9% or 99.5% or99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or 95.5% or95% or 94.5% complementarity between the sequence and the guide.

In certain embodiments, modulations of cleavage efficiency can beexploited by introduction of mismatches, e.g. 1 or more mismatches, suchas 1 or 2 mismatches between spacer sequence and target sequence,including the position of the mismatch along the spacer/target. The morecentral (i.e. not 3′ or 5′) for instance a double mismatch is, the morecleavage efficiency is affected. Accordingly, by chosing mismatchposition along the spacer, cleavage efficiency can be modulated. Bymeans of example, if less than 100% cleavage of targets is desired (e.g.in a cell population), 1 or more, such as preferably 2 mismatchesbetween spacer and target sequence may be introduced in the spacersequences. The more central along the spacer of the mismatch position,the lower the cleavage percentage.

The methods according to the invention as described herein comprehendinducing one or more nucleotide modifications in a eukaryotic cell (invitro, i.e. in an isolated eukaryotic cell) as herein discussedcomprising delivering to cell a vector as herein discussed. Themutation(s) can include the introduction, deletion, or substitution ofone or more nucleotides at each target sequence of cell(s) via theguide(s) RNA(s) or sgRNA(s). The mutations can include the introduction,deletion, or substitution of 1-75 nucleotides at each target sequence ofsaid cell(s) via the guide(s) RNA(s). The mutations can include theintroduction, deletion, or substitution of 1, 5, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45,50, or 75 nucleotides at each target sequence of said cell(s) via theguide(s) RNA(s). The mutations can include the introduction, deletion,or substitution of 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides ateach target sequence of said cell(s) via the guide(s) RNA(s). Themutations include the introduction, deletion, or substitution of 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 35, 40, 45, 50, or 75 nucleotides at each target sequence of saidcell(s) via the guide(s) RNA(s). The mutations can include theintroduction, deletion, or substitution of 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at each targetsequence of said cell(s) via the guide(s) RNA(s). The mutations caninclude the introduction, deletion, or substitution of 40, 45, 50, 75,100, 200, 300, 400 or 500 nucleotides at each target sequence of saidcell(s) via the guide(s) RNA(s).

For minimization of toxicity and off-target effect, it will be importantto control the concentration of Cas mRNA or protein and guide RNAdelivered. Optimal concentrations of Cas mRNA or protein and guide RNAcan be determined by testing different concentrations in a cellular ornon-human eukaryote animal model and using deep sequencing the analyzethe extent of modification at potential off-target genomic loci.

Typically, in the context of an endogenous CRISPR system, formation of aCRISPR complex (comprising a guide sequence hybridized to a targetsequence and complexed with one or more Cas proteins) results incleavage in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50,or more base pairs from) the target sequence, but may depend on forinstance secondary structure, in particular in the case of RNA targets.

The nucleic acid molecule encoding a Cas is advantageously codonoptimized Cas. An example of a codon optimized sequence, is in thisinstance a sequence optimized for expression in a eukaryote, e.g.,humans (i.e. being optimized for expression in humans), or for anothereukaryote, animal or mammal as herein discussed; see, e.g., SaCas9 humancodon optimized sequence in WO 2014/093622 (PCT/US2013/074667). Whilstthis is preferred, it will be appreciated that other examples arepossible and codon optimization for a host species other than human, orfor codon optimization for specific organs is known. In someembodiments, an enzyme coding sequence encoding a Cas is codon optimizedfor expression in particular cells, such as eukaryotic cells. Theeukaryotic cells may be those of or derived from a particular organism,such as a mammal, including but not limited to human, or non-humaneukaryote or animal or mammal as herein discussed, e.g., mouse, rat,rabbit, dog, livestock, or non-human mammal or primate. In someembodiments, processes for modifying the germ line genetic identity ofhuman beings and/or processes for modifying the genetic identity ofanimals which are likely to cause them suffering without any substantialmedical benefit to man or animal, and also animals resulting from suchprocesses, may be excluded. In general, codon optimization refers to aprocess of modifying a nucleic acid sequence for enhanced expression inthe host cells of interest by replacing at least one codon (e.g. aboutor more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) ofthe native sequence with codons that are more frequently or mostfrequently used in the genes of that host cell while maintaining thenative amino acid sequence. Various species exhibit particular bias forcertain codons of a particular amino acid. Codon bias (differences incodon usage between organisms) often correlates with the efficiency oftranslation of messenger RNA (mRNA), which is in turn believed to bedependent on, among other things, the properties of the codons beingtranslated and the availability of particular transfer RNA (tRNA)molecules. The predominance of selected tRNAs in a cell is generally areflection of the codons used most frequently in peptide synthesis.Accordingly, genes can be tailored for optimal gene expression in agiven organism based on codon optimization. Codon usage tables arereadily available, for example, at the “Codon Usage Database” availableat www.kazusa.orjp/codon/and these tables can be adapted in a number ofways. See Nakamura, Y., et al. “Codon usage tabulated from theinternational DNA sequence databases: status for the year 2000” Nucl.Acids Res. 28:292 (2000). Computer algorithms for codon optimizing aparticular sequence for expression in a particular host cell are alsoavailable, such as Gene Forge (Aptagen; Jacobus, Pa.), are alsoavailable. In some embodiments, one or more codons (e.g. 1, 2, 3, 4, 5,10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a Cascorrespond to the most frequently used codon for a particular aminoacid.

In certain embodiments, the methods as described herein may compriseproviding a Cas transgenic cell in which one or more nucleic acidsencoding one or more guide RNAs are provided or introduced operablyconnected in the cell with a regulatory element comprising a promoter ofone or more gene of interest. As used herein, the term “Cas transgeniccell” refers to a cell, such as a eukaryotic cell, in which a Cas genehas been genomically integrated. The nature, type, or origin of the cellare not particularly limiting according to the present invention. Alsothe way how the Cas transgene is introduced in the cell is may vary andcan be any method as is known in the art. In certain embodiments, theCas transgenic cell is obtained by introducing the Cas transgene in anisolated cell. In certain other embodiments, the Cas transgenic cell isobtained by isolating cells from a Cas transgenic organism. By means ofexample, and without limitation, the Cas transgenic cell as referred toherein may be derived from a Cas transgenic eukaryote, such as a Casknock-in eukaryote. Reference is made to WO 2014/093622(PCT/US13/74667), incorporated herein by reference. Methods of US PatentPublication Nos. 20120017290 and 20110265198 assigned to SangamoBioSciences, Inc. directed to targeting the Rosa locus may be modifiedto utilize the CRISPR Cas system of the present invention. Methods of USPatent Publication No. 20130236946 assigned to Cellectis directed totargeting the Rosa locus may also be modified to utilize the CRISPR Cassystem of the present invention. By means of further example referenceis made to Platt et. al. (Cell; 159(2):440-455 (2014)), describing aCas9 knock-in mouse, which is incorporated herein by reference. The Castransgene can further comprise a Lox-Stop-polyA-Lox(LSL) cassettethereby rendering Cas expression inducible by Cre recombinase.Alternatively, the Cas transgenic cell may be obtained by introducingthe Cas transgene in an isolated cell. Delivery systems for transgenesare well known in the art. By means of example, the Cas transgene may bedelivered in for instance eukaryotic cell by means of vector (e.g., AAV,adenovirus, lentivirus) and/or particle and/or nanoparticle delivery, asalso described herein elsewhere.

It will be understood by the skilled person that the cell, such as theCas transgenic cell, as referred to herein may comprise further genomicalterations besides having an integrated Cas gene or the mutationsarising from the sequence specific action of Cas when complexed with RNAcapable of guiding Cas to a target locus, such as for instance one ormore oncogenic mutations, as for instance and without limitationdescribed in Platt et al. (2014), Chen et al., (2014) or Kumar et al.(2009).

In some embodiments, the Cas sequence is fused to one or more nuclearlocalization sequences (NLSs) or nuclear export signals (NESs), such asabout or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs orNESs. In some embodiments, the Cas comprises about or more than about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs or NESs at or near theamino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more NLSs or NESs at or near the carboxy-terminus, or a combinationof these (e.g. zero or at least one or more NLS or NES at theamino-terminus and zero or at one or more NLS or NES at the carboxyterminus). When more than one NLS or NES is present, each may beselected independently of the others, such that a single NLS or NES maybe present in more than one copy and/or in combination with one or moreother NLSs or NESs present in one or more copies. In a preferredembodiment of the invention, the Cas comprises at most 6 NLSs. In someembodiments, an NLS or NES is considered near the N- or C-terminus whenthe nearest amino acid of the NLS or NES is within about 1, 2, 3, 4, 5,10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptidechain from the N- or C-terminus. Non-limiting examples of NLSs includean NLS sequence derived from: the NLS of the SV40 virus large T-antigen,having the amino acid sequence PKKKRKV (SEQ ID NO: 100); the NLS fromnucleoplasmin (e.g. the nucleoplasmin bipartite NLS with the sequenceKRPAATKKAGQAKKKK) (SEQ ID NO:101); the c-myc NLS having the amino acidsequence PAAKRVKLD (SEQ ID NO: 102) or RQRRNELKRSP (SEQ ID NO:103); thehRNPA1 M9 NLS having the sequenceNQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY(SEQ ID NO: 104); the sequenceRMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 105) of the IBBdomain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 106) andPPKKARED (SEQ ID NO: 107) of the myoma T protein; the sequence POPKKKPL(SEQ ID NO: 108) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO:109) of mouse c-ab1 IV; the sequences DRLRR (SEQ ID NO: 110) and PKQKKRK(SEQ ID NO: 111) of the influenza virus NS1; the sequence RKLKKKIKKL(SEQ ID NO: 112) of the Hepatitis virus delta antigen; the sequenceREKKKFLKRR (SEQ ID NO: 113) of the mouse Mx1 protein; the sequenceKRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 114) of the human poly(ADP-ribose)polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 115) of thesteroid hormone receptors (human) glucocorticoid. Non-limiting examplesof NESs include an NES sequence LYPERLRRILT (SEQ ID NO: 116)(ctgtaccctgagcggctgcggcggatcctgacc (SEQ ID NO: 117)). In general, theone or more NLSs or NESs are of sufficient strength to driveaccumulation of the Cas in a detectable amount in respectively thenucleus or the cytoplasm of a eukaryotic cell. In general, strength ofnuclear localization/export activity may derive from the number ofNLSs/NESs in the Cas, the particular NLS(s) or NES(s) used, or acombination of these factors. Detection of accumulation in thenucleus/cytoplasm may be performed by any suitable technique. Forexample, a detectable marker may be fused to the Cas, such that locationwithin a cell may be visualized, such as in combination with a means fordetecting the location of the nucleus (e.g. a stain specific for thenucleus such as DAPI) or cytoplasm. Cell nuclei may also be isolatedfrom cells, the contents of which may then be analyzed by any suitableprocess for detecting protein, such as immunohistochemistry, Westernblot, or enzyme activity assay. Accumulation in the nucleus may also bedetermined indirectly, such as by an assay for the effect of CRISPRcomplex formation (e.g. assay for DNA cleavage or mutation at the targetsequence, or assay for altered gene expression activity affected byCRISPR complex formation and/or Cas enzyme activity), as compared to acontrol no exposed to the Cas or complex, or exposed to a Cas lackingthe one or more NLSs or NESs. In certain embodiments, other localizationtags may be fused to the Cas protein, such as without limitation forlocalizing the Cas to particular sites in a cell, such as organells,such mitochondria, plastids, chloroplast, vesicles, golgi, (nuclear orcellular) membranes, ribosomes, nucleoluse, ER, cytoskeleton, vacuoles,centrosome, nucleosome, granules, centrioles, etc.

In certain aspects the invention involves vectors, e.g. for deliveringor introducing in a cell Cas and/or RNA capable of guiding Cas to atarget locus (i.e. guide RNA), but also for propagating these components(e.g. in prokaryotic cells). A used herein, a “vector” is a tool thatallows or facilitates the transfer of an entity from one environment toanother. It is a replicon, such as a plasmid, phage, or cosmid, intowhich another DNA segment may be inserted so as to bring about thereplication of the inserted segment. Generally, a vector is capable ofreplication when associated with the proper control elements. Ingeneral, the term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. Vectorsinclude, but are not limited to, nucleic acid molecules that aresingle-stranded, double-stranded, or partially double-stranded; nucleicacid molecules that comprise one or more free ends, no free ends (e.g.circular); nucleic acid molecules that comprise DNA, RNA, or both; andother varieties of polynucleotides known in the art. One type of vectoris a “plasmid,” which refers to a circular double stranded DNA loop intowhich additional DNA segments can be inserted, such as by standardmolecular cloning techniques. Another type of vector is a viral vector,wherein virally-derived DNA or RNA sequences are present in the vectorfor packaging into a virus (e.g. retroviruses, replication defectiveretroviruses, adenoviruses, replication defective adenoviruses, andadeno-associated viruses (AAVs)). Viral vectors also includepolynucleotides carried by a virus for transfection into a host cell.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g. bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively-linked. Such vectors are referred to herein as “expressionvectors.” Common expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell). With regards torecombination and cloning methods, mention is made of U.S. patentapplication Ser. No. 10/815,730, published Sep. 2, 2004 as US2004-0171156 A1, the contents of which are herein incorporated byreference in their entirety.

The vector(s) can include the regulatory element(s), e.g., promoter(s).The vector(s) can comprise Cas encoding sequences, and/or a single, butpossibly also can comprise at least 3 or 8 or 16 or 32 or 48 or 50 guideRNA(s) (e.g., sgRNAs) encoding sequences, such as 1-2, 1-3, 1-4 1-5,3-6, 3-7, 3-8, 3-9, 3-10, 3-8, 3-16, 3-30, 3-32, 3-48, 3-50 RNA(s)(e.g., sgRNAs). In a single vector there can be a promoter for each RNA(e.g., sgRNA), advantageously when there are up to about 16 RNA(s); and,when a single vector provides for more than 16 RNA(s), one or morepromoter(s) can drive expression of more than one of the RNA(s), e.g.,when there are 32 RNA(s), each promoter can drive expression of twoRNA(s), and when there are 48 RNA(s), each promoter can drive expressionof three RNA(s). By simple arithmetic and well established cloningprotocols and the teachings in this disclosure one skilled in the artcan readily practice the invention as to the RNA(s) for a suitableexemplary vector such as AAV, and a suitable promoter such as the U6promoter. For example, the packaging limit of AAV is ˜4.7 kb. The lengthof a single U6-gRNA (plus restriction sites for cloning) is 361 bp.Therefore, the skilled person can readily fit about 12-16, e.g., 13U6-gRNA cassettes in a single vector. This can be assembled by anysuitable means, such as a golden gate strategy used for TALE assembly(http://www.genome-engineering.org/taleffectors/). The skilled personcan also use a tandem guide strategy to increase the number of U6-gRNAsby approximately 1.5 times, e.g., to increase from 12-16, e.g., 13 toapproximately 18-24, e.g., about 19 U6-gRNAs. Therefore, one skilled inthe art can readily reach approximately 18-24, e.g., about 19promoter-RNAs, e.g., U6-gRNAs in a single vector, e.g., an AAV vector. Afurther means for increasing the number of promoters and RNAs in avector is to use a single promoter (e.g., U6) to express an array ofRNAs separated by cleavable sequences. And an even further means forincreasing the number of promoter-RNAs in a vector, is to express anarray of promoter-RNAs separated by cleavable sequences in the intron ofa coding sequence or gene; and, in this instance it is advantageous touse a polymerase II promoter, which can have increased expression andenable the transcription of long RNA in a tissue specific manner. (see,e.g., http://nar.oxfordjournals.org/content/34/7/e53.short,http://www.nature.com/mt/journal/v16/n9/abs/mt2008144a.html). In anadvantageous embodiment, AAV may package U6 tandem gRNA targeting up toabout 50 genes. Accordingly, from the knowledge in the art and theteachings in this disclosure the skilled person can readily make and usevector(s), e.g., a single vector, expressing multiple RNAs or guidesunder the control or operatively or functionally linked to one or morepromoters—especially as to the numbers of RNAs or guides discussedherein, without any undue experimentation.

The guide RNA(s) encoding sequences and/or Cas encoding sequences, canbe functionally or operatively linked to regulatory element(s) and hencethe regulatory element(s) drive expression. The promoter(s) can beconstitutive promoter(s) and/or conditional promoter(s) and/or induciblepromoter(s) and/or tissue specific promoter(s). The promoter can beselected from the group consisting of RNA polymerases, pol I, pol II,pol III, T7, U6, H1, retroviral Rous sarcoma virus (RSV) LTR promoter,the cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolatereductase promoter, the β-actin promoter, the phosphoglycerol kinase(PGK) promoter, and the EF1α promoter. An advantageous promoter is thepromoter is U6.

Aspects of the invention relate to the identification and engineering ofnovel effector proteins associated with Class 2 CRISPR-Cas systems. In apreferred embodiment, the effector protein comprises a single-subuniteffector module. In a further embodiment the effector protein isfunctional in prokaryotic or eukaryotic cells for in vitro, in vivo orex vivo applications.

The term “nucleic acid-targeting system”, wherein nucleic acid is DNA orRNA, and in some aspects may also refer to DNA-RNA hybrids orderivatives thereof, refers collectively to transcripts and otherelements involved in the expression of or directing the activity of DNAor RNA-targeting CRISPR-associated (“Cas”) genes, which may includesequences encoding a DNA or RNA-targeting Cas protein and a DNA orRNA-targeting guide RNA comprising a CRISPR RNA (crRNA) sequence and (insome but not all systems) a trans-activating CRISPR/Cas system RNA(tracrRNA) sequence, or other sequences and transcripts from a DNA orRNA-targeting CRISPR locus. In general, a RNA-targeting system ischaracterized by elements that promote the formation of a DNA orRNA-targeting complex at the site of a target DNA or RNA sequence. Inthe context of formation of a DNA or RNA-targeting complex, “targetsequence” refers to a DNA or RNA sequence to which a DNA orRNA-targeting guide RNA is designed to have complementarity, wherehybridization between a target sequence and a RNA-targeting guide RNApromotes the formation of a RNA-targeting complex. In some embodiments,a target sequence is located in the nucleus or cytoplasm of a cell.

In an aspect of the invention, novel RNA targeting systems also referredto as RNA- or RNA-targeting CRISPR/Cas or the CRISPR-Cas systemRNA-targeting system of the present application are based on identifiedType VI-B Cas proteins which do not require the generation of customizedproteins to target specific RNA sequences but rather a single enzyme canbe programmed by a RNA molecule to recognize a specific RNA target, inother words the enzyme can be recruited to a specific RNA target usingsaid RNA molecule.

In an aspect of the invention, novel DNA targeting systems also referredto as DNA- or DNA-targeting CRISPR/Cas or the CRISPR-Cas systemRNA-targeting system of the present application are based on identifiedType VI-B Cas proteins which do not require the generation of customizedproteins to target specific RNA sequences but rather a single enzyme canbe programmed by a RNA molecule to recognize a specific DNA target, inother words the enzyme can be recruited to a specific DNA target usingsaid RNA molecule.

The nucleic acids-targeting systems, the vector systems, the vectors andthe compositions described herein may be used in various nucleicacids-targeting applications, altering or modifying synthesis of a geneproduct, such as a protein, nucleic acids cleavage, nucleic acidsediting, nucleic acids splicing; trafficking of target nucleic acids,tracing of target nucleic acids, isolation of target nucleic acids,visualization of target nucleic acids, etc.

As used herein, a Cas protein or a CRISPR enzyme refers to any of theproteins presented in the new classification of CRISPR-Cas systems.

Cas13b Nucleases

The Cas13b effector protein of the invention is, or comprises, orconsists essentially of, or consists of, or involves or relates to sucha protein from or as set forth in FIG. 1. Preferred proteins of FIG. 1are selected from the group consisting of Porphyromonas gulae Cas13b(accession number WP_039434803), Prevotella sp. P5-125 Cas13b (accessionnumber WP_044065294), Porphyromonas gingivalis Cas13b (accession numberWP_053444417), Porphyromonas sp. COT-052 OH4946 Cas13b (accession numberWP_039428968), Bacteroides pyogenes Cas13b (accession numberWP_034542281), Riemerella anatipestifer Cas13b (accession numberWP_004919755). The most preferred proteins of FIG. 1 are selected fromthe group consisting of Porphyromonas gulae Cas13b (accession numberWP_039434803), Prevotella sp. P5-125 Cas13b (accession numberWP_044065294), Porphyromonas gingivalis Cas13b (accession numberWP_053444417), Porphyromonas sp. COT-052 OH4946 Cas13b (accession numberWP_039428968); and most specifically preferred are Porphyromonas gulaeCas13b (accession number WP_039434803) or Prevotella sp. P5-125 Cas13b(accession number WP_044065294). This invention is intended to provide,or relate to, or involve, or comprise, or consist essentially of, orconsist of, a protein from or as set forth herein, including mutationsor alterations thereof as set forth herein

Thus, in some embodiments, the effector protein may be a RNA-bindingprotein, such as a dead-Cas type effector protein, which may beoptionally functionalised as described herein for instance with antranscriptional activator or repressor domain, NLS or other functionaldomain. In some embodiments, the effector protein may be a RNA-bindingprotein that cleaves a single strand of RNA. If the RNA bound is ssRNA,then the ssRNA is fully cleaved. In some embodiments, the effectorprotein may be a RNA-binding protein that cleaves a double strand ofRNA, for example if it comprises two RNase domains. If the RNA bound isdsRNA, then the dsRNA is fully cleaved.

RNase function in CRISPR systems is known, for example mRNA targetinghas been reported for certain type III CRISPR-Cas systems (Hale et al.,2014, Genes Dev, vol. 28, 2432-2443; Hale et al., 2009, Cell, vol. 139,945-956; Peng et al., 2015, Nucleic acids research, vol. 43, 406-417)and provides significant advantages. A CRISPR-Cas system, composition ormethod targeting RNA via the present effector proteins is thus provided.

The target RNA, i.e. the RNA of interest, is the RNA to be targeted bythe present invention leading to the recruitment to, and the binding ofthe effector protein at, the target site of interest on the target RNA.The target RNA may be any suitable form of RNA. This may include, insome embodiments, mRNA. In other embodiments, the target RNA may includetRNA or rRNA.

Cas13b Guide

As used herein, the term “crRNA” or “guide RNA” or “single guide RNA” or“sgRNA” or “one or more nucleic acid components” of a Type VI CRISPR-Caslocus effector protein comprises any polynucleotide sequence havingsufficient complementarity with a target nucleic acid sequence tohybridize with the target nucleic acid sequence and directsequence-specific binding of a RNA-targeting complex to the target RNAsequence.

In certain embodiments, the CRISPR system as provided herein can makeuse of a crRNA or analogous polynucleotide comprising a guide sequence,wherein the polynucleotide is an RNA, a DNA or a mixture of RNA and DNA,and/or wherein the polynucleotide comprises one or more nucleotideanalogs. The sequence can comprise any structure, including but notlimited to a structure of a native crRNA, such as a bulge, a hairpin ora stem loop structure. In certain embodiments, the polynucleotidecomprising the guide sequence forms a duplex with a secondpolynucleotide sequence which can be an RNA or a DNA sequence.

In certain embodiments, guides of the invention comprise non-naturallyoccurring nucleic acids and/or non-naturally occurring nucleotidesand/or nucleotide analogs, and/or chemically modifications.Non-naturally occurring nucleic acids can include, for example, mixturesof naturally and non-naturally occurring nucleotides. Non-naturallyoccurring nucleotides and/or nucleotide analogs may be modified at theribose, phosphate, and/or base moiety. In an embodiment of theinvention, a guide nucleic acid comprises ribonucleotides andnon-ribonucleotides. In one such embodiment, a guide comprises one ormore ribonucleotides and one or more deoxyribonucleotides. In anembodiment of the invention, the guide comprises one or morenon-naturally occurring nucleotide or nucleotide analog such as anucleotide with phosphorothioate linkage, boranophosphate linkage, alocked nucleic acid (LNA) nucleotides comprising a methylene bridgebetween the 2′ and 4′ carbons of the ribose ring, or bridged nucleicacids (BNA). Other examples of modified nucleotides include 2′-O-methylanalogs, 2′-deoxy analogs, 2-thiouridine analogs, N6-methyladenosineanalogs, or 2′-fluoro analogs. Further examples of modified basesinclude, but are not limited to, 2-aminopurine, 5-bromo-uridine,pseudouridine (Ψ), N1-methylpseudouridine (me1Ψ), 5-methoxyuridine(5moU), inosine, 7-methylguanosine. Examples of guide RNA chemicalmodifications include, without limitation, incorporation of 2′-O-methyl(M), 2′-O-methyl 3′phosphorothioate (MS), S-constrained ethyl (cEt), or2′-O-methyl 3′thioPACE (MSP) at one or more terminal nucleotides. Suchchemically modified guide RNAs can comprise increased stability andincreased activity as compared to unmodified guide RNAs, thoughon-target vs. off-target specificity is not predictable. (See, Hendel,2015, Nat Biotechnol. 33(9):985-9, doi: 10.1038/nbt.3290, publishedonline 29 Jun. 2015; Allerson et al., J. Med. Chem. 2005, 48:901-904;Bramsen et al., Front. Genet., 2012, 3:154; Deng et al., PNAS, 2015,112:11870-11875; Sharma et al., MedChemComm., 2014, 5:1454-1471; Li etal., Nature Biomedical Engineering, 2017, 1, 0066DOI:10.1038/s41551-017-0066).

In some embodiments, the 5′ and/or 3′ end of a guide RNA is modified bya variety of functional moieties including fluorescent dyes,polyethylene glycol, cholesterol, proteins, or detection tags. (SeeKelly et al., 2016, J. Biotech. 233:74-83). In certain embodiments, aguide comprises ribonucleotides in a region that binds to a target RNAand one or more deoxyribonucleotides and/or nucleotide analogs in aregion that binds to Cas13b. In an embodiment of the invention,deoxyribonucleotides and/or nucleotide analogs are incorporated inengineered guide structures, such as, without limitation, 5′ and/or 3′end, stem-loop regions, and the seed region. In certain embodiments, themodification is not in the 3′-handle of the stem-loop regions. Chemicalmodification in the 3′-handle of the stem-loop region of a guide mayabolish its function. In certain embodiments, at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides of a guide ischemically modified. In some embodiments, 3-5 nucleotides at either the3′ or the 5′ end of a guide is chemically modified. In some embodiments,only minor modifications are introduced in the seed region, such as 2′-Fmodifications. In some embodiments, 2′-F modification is introduced atthe 5′ and/or the 3′ end of a guide. In certain embodiments, three tofive nucleotides at the 5′ and/or the 3′ end of the guide are chemicallymodified with 2′-O-methyl (M), 2′-O-methyl-3′-phosphorothioate (MS),S-constrained ethyl(cEt), or 2′-O-methyl-3′-thioPACE (MSP). Suchmodification can enhance genome editing efficiency (see Hendel et al.,Nat. Biotechnol. (2015) 33(9): 985-989). In certain embodiments, all ofthe phosphodiester bonds of a guide are substituted withphosphorothioates (PS) for enhancing levels of gene disruption. Incertain embodiments, more than five nucleotides at the 5′ and/or the 3′end of the guide are chemically modified with 2′-O-Me, 2′-F orS-constrained ethyl(cEt). Such chemically modified guide can mediateenhanced levels of gene disruption (see Ragdarm et al., 0215, PNAS,E7110-E7111). In an embodiment of the invention, a guide is modified tocomprise a chemical moiety at its 3′ and/or 5′ end. Such moietiesinclude, but are not limited to amine, azide, alkyne, thio,dibenzocyclooctyne (DBCO), or Rhodamine. In certain embodiment, thechemical moiety is conjugated to the guide by a linker, such as an alkylchain. In certain embodiments, the chemical moiety of the modified guidecan be used to attach the guide to another molecule, such as DNA, RNA,protein, or nanoparticles. Such chemically modified guide can be used toidentify or enrich cells generically edited by a CRISPR system (see Leeet al., eLife, 2017, 6:e25312, DOI:10.7554)

In some embodiments, the modification to the guide is a chemicalmodification, an insertion, a deletion or a split. In some embodiments,the chemical modification includes, but is not limited to, incorporationof 2′-O-methyl (M) analogs, 2′-deoxy analogs, 2-thiouridine analogs,N6-methyladenosine analogs, 2′-fluoro analogs, 2-aminopurine,5-bromo-uridine, pseudouridine (T), N1-methylpseudouridine (mePP),5-methoxyuridine (5moU), inosine, 7-methylguanosine,2′-O-methyl-3′-phosphorothioate (MS), S-constrained ethyl(cEt),phosphorothioate (PS), or 2′-O-methyl-3′-thioPACE (MSP). In someembodiments, the guide comprises one or more of phosphorothioatemodifications. In certain embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 nucleotides of theguide are chemically modified. In certain embodiments, one or morenucleotides in the seed region are chemically modified. In certainembodiments, one or more nucleotides in the 5′-terminus are chemicallymodified. In certain embodiments, none of the nucleotides in the3′-handle is chemically modified. In some embodiments, the chemicalmodification in the seed region is a minor modification, such asincorporation of a 2′-fluoro analog. In a specific embodiment, onenucleotide of the seed region is replaced with a 2′-fluoro analog. Insome embodiments, 5 or 10 nucleotides in the 5′-terminus are chemicallymodified. Such chemical modifications at the 5′-terminus of the Cas13bCrRNA may improve gene cutting efficiency. In a specific embodiment, 5nucleotides in the 5′-terminus are replaced with 2′-fluoro analogues. Ina specific embodiment, 10 nucleotides in the 5′-terminus are replacedwith 2′-fluoro analogues. In a specific embodiment, 5 nucleotides in the5′-terminus are replaced with 2′-O-methyl (M) analogs.

In some embodiments, the loop of the 3′-handle of the guide is modified.In some embodiments, the loop of the 3′-handle of the guide is modifiedto have a deletion, an insertion, a split, or chemical modifications. Incertain embodiments, the loop comprises 3, 4, or 5 nucleotides. Incertain embodiments, the loop comprises the sequence of UCUU, UUUU,UAUU, or UGUU.

In one aspect, the guide comprises portions that are chemically linkedor conjugated via a non-phosphodiester bond. In one aspect, the guidecomprises, in non-limiting examples, a direct repeat and a targetingsequence portion that are chemically linked or conjugated via anon-nucleotide loop. In some embodiments, the portions are joined via anon-phosphodiester covalent linker. Examples of the covalent linkerinclude but are not limited to a chemical moiety selected from the groupconsisting of carbamates, ethers, esters, amides, imines, amidines,aminotrizines, hydrozone, disulfides, thioethers, thioesters,phosphorothioates, phosphorodithioates, sulfonamides, sulfonates,fulfones, sulfoxides, ureas, thioureas, hydrazide, oxime, triazole,photolabile linkages, C—C bond forming groups such as Diels-Aldercyclo-addition pairs or ring-closing metathesis pairs, and Michaelreaction pairs.

In some embodiments, portions of the guide are first synthesized usingthe standard phosphoramidite synthetic protocol (Herdewijn, P., ed.,Methods in Molecular Biology Col 288, Oligonucleotide Synthesis: Methodsand Applications, Humana Press, New Jersey (2012)). In some embodiments,the non-targeting guide portions can be functionalized to contain anappropriate functional group for ligation using the standard protocolknown in the art (Hermanson, G. T., Bioconjugate Techniques, AcademicPress (2013)). Examples of functional groups include, but are notlimited to, hydroxyl, amine, carboxylic acid, carboxylic acid halide,carboxylic acid active ester, aldehyde, carbonyl, chlorocarbonyl,imidazolylcarbonyl, hydrozide, semicarbazide, thio semicarbazide, thiol,maleimide, haloalkyl, sulfonyl, ally, propargyl, diene, alkyne, andazide. Once a non-targeting portions of a guide is functionalized, acovalent chemical bond or linkage can be formed between the twooligonucleotides. Examples of chemical bonds include, but are notlimited to, those based on carbamates, ethers, esters, amides, imines,amidines, aminotrizines, hydrozone, disulfides, thioethers, thioesters,phosphorothioates, phosphorodithioates, sulfonamides, sulfonates,fulfones, sulfoxides, ureas, thioureas, hydrazide, oxime, triazole,photolabile linkages, C—C bond forming groups such as Diels-Aldercyclo-addition pairs or ring-closing metathesis pairs, and Michaelreaction pairs.

In some embodiments, one or more portions of a guide can be chemicallysynthesized. In some embodiments, the chemical synthesis uses automated,solid-phase oligonucleotide synthesis machines with 2′-acetoxyethylorthoester (2′-ACE) (Scaringe et al., J. Am. Chem. Soc. (1998) 120:11820-11821; Scaringe, Methods Enzymol. (2000) 317: 3-18) or2′-thionocarbamate (2′-TC) chemistry (Dellinger et al., J. Am. Chem.Soc. (2011) 133: 11540-11546; Hendel et al., Nat. Biotechnol. (2015)33:985-989).

In some embodiments, the guide portions can be covalently linked usingvarious bioconjugation reactions, loops, bridges, and non-nucleotidelinks via modifications of sugar, internucleotide phosphodiester bonds,purine and pyrimidine residues. Sletten et al., Angew. Chem. Int. Ed.(2009) 48:6974-6998; Manoharan, M. Curr. Opin. Chem. Biol. (2004) 8:570-9; Behlke et al., Oligonucleotides (2008) 18: 305-19; Watts, et al.,Drug. Discov. Today (2008) 13: 842-55; Shukla, et al., ChemMedChem(2010) 5: 328-49.

In some embodiments, the guide portions can be covalently linked usingclick chemistry. In some embodiments, guide portions can be covalentlylinked using a triazole linker. In some embodiments, guide portions canbe covalently linked using Huisgen 1,3-dipolar cycloaddition reactioninvolving an alkyne and azide to yield a highly stable triazole linker(He et al., ChemBioChem (2015) 17: 1809-1812; WO 2016/186745). In someembodiments, guide portions are covalently linked by ligating a5′-hexyne portion and a 3′-azide portion. In some embodiments, either orboth of the 5′-hexyne guide portion and a 3′-azide guide portion can beprotected with 2′-acetoxyethyl orthoester (2′-ACE) group, which can besubsequently removed using Dharmacon protocol (Scaringe et al., J. Am.Chem. Soc. (1998) 120: 11820-11821; Scaringe, Methods Enzymol. (2000)317: 3-18).

In some embodiments, guide portions can be covalently linked via alinker (e.g., a non-nucleotide loop) that comprises a moiety such asspacers, attachments, bioconjugates, chromophores, reporter groups, dyelabeled RNAs, and non-naturally occurring nucleotide analogues. Morespecifically, suitable spacers for purposes of this invention include,but are not limited to, polyethers (e.g., polyethylene glycols,polyalcohols, polypropylene glycol or mixtures of ethylene and propyleneglycols), polyamines group (e.g., spennine, spermidine and polymericderivatives thereof), polyesters (e.g., poly(ethyl acrylate)),polyphosphodiesters, alkylenes, and combinations thereof. Suitableattachments include any moiety that can be added to the linker to addadditional properties to the linker, such as but not limited to,fluorescent labels. Suitable bioconjugates include, but are not limitedto, peptides, glycosides, lipids, cholesterol, phospholipids, diacylglycerols and dialkyl glycerols, fatty acids, hydrocarbons, enzymesubstrates, steroids, biotin, digoxigenin, carbohydrates,polysaccharides. Suitable chromophores, reporter groups, and dye-labeledRNAs include, but are not limited to, fluorescent dyes such asfluorescein and rhodamine, chemiluminescent, electrochemiluminescent,and bioluminescent marker compounds. The design of example linkersconjugating two RNA components are also described in WO 2004/015075.

The linker (e.g., a non-nucleotide loop) can be of any length. In someembodiments, the linker has a length equivalent to about 0-16nucleotides. In some embodiments, the linker has a length equivalent toabout 0-8 nucleotides. In some embodiments, the linker has a lengthequivalent to about 0-4 nucleotides. In some embodiments, the linker hasa length equivalent to about 2 nucleotides. Example linker design isalso described in WO2011/008730.

In some embodiments, the degree of complementarity, when optimallyaligned using a suitable alignment algorithm, is about or more thanabout 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimalalignment may be determined with the use of any suitable algorithm foraligning sequences, non-limiting example of which include theSmith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithmsbased on the Burrows-Wheeler Transform (e.g., the Burrows WheelerAligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies;available at www.novocraft.com), ELAND (Illumina, San Diego, Calif.),SOAP (available at soap.genomics.org.cn), and Maq (available atmaq.sourceforge.net). The ability of a guide sequence (within aRNA-targeting guide RNA or crRNA) to direct sequence-specific binding ofa nucleic acid-targeting complex to a target nucleic acid sequence maybe assessed by any suitable assay. For example, the components of aRNA-targeting CRISPR Cas13b system sufficient to form a nucleicacid-targeting complex, including the guide sequence to be tested, maybe provided to a host cell having the corresponding target nucleic acidsequence, such as by transfection with vectors encoding the componentsof the nucleic acid-targeting complex, followed by an assessment ofpreferential targeting (e.g., cleavage) within the target nucleic acidsequence, such as by Surveyor assay as described herein. Similarly,cleavage of a target nucleic acid sequence may be evaluated in a testtube by providing the target nucleic acid sequence, components of anucleic acid-targeting complex, including the guide sequence to betested and a control guide sequence different from the test guidesequence, and comparing binding or rate of cleavage at the targetsequence between the test and control guide sequence reactions. Otherassays are possible, and will occur to those skilled in the art. A guidesequence, and hence a RNA-targeting guide RNA or crRNA may be selectedto target any target nucleic acid sequence. The target sequence may beDNA. The target sequence may be any RNA sequence. In some embodiments,the target sequence may be a sequence within a RNA molecule selectedfrom the group consisting of messenger RNA (mRNA), pre-mRNA, ribosomalRNA (rRNA), transfer RNA (tRNA), micro-RNA (miRNA), small interferingRNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA),double stranded RNA (dsRNA), non coding RNA (ncRNA), long non-coding RNA(lncRNA), and small cytoplasmatic RNA (scRNA). In some preferredembodiments, the target sequence may be a sequence within a RNA moleculeselected from the group consisting of mRNA, pre-mRNA, and rRNA. In somepreferred embodiments, the target sequence may be a sequence within aRNA molecule selected from the group consisting of ncRNA, and lncRNA. Insome more preferred embodiments, the target sequence may be a sequencewithin an mRNA molecule or a pre-mRNA molecule.

In some embodiments, a RNA-targeting guide RNA or crRNA is selected toreduce the degree secondary structure within the RNA-targeting guide RNAor crRNA. In some embodiments, about or less than about 75%, 50%, 40%,30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of theRNA-targeting guide RNA participate in self-complementary base pairingwhen optimally folded. Optimal folding may be determined by any suitablepolynucleotide folding algorithm. Some programs are based on calculatingthe minimal Gibbs free energy. An example of one such algorithm ismFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981),133-148). Another example folding algorithm is the online webserverRNAfold, developed at Institute for Theoretical Chemistry at theUniversity of Vienna, using the centroid structure prediction algorithm(see e.g., A. R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carrand GM Church, 2009, Nature Biotechnology 27(12): 1151-62).

In certain embodiments, a guide RNA or crRNA may comprise, consistessentially of, or consist of a direct repeat (DR) sequence and a guidesequence or spacer sequence. In certain embodiments, the guide RNA orcrRNA may comprise, consist essentially of, or consist of a directrepeat sequence fused or linked to a guide sequence or spacer sequence.In certain embodiments, the direct repeat sequence may be locatedupstream (i.e., 5′) from the guide sequence or spacer sequence. In otherembodiments, the direct repeat sequence may be located downstream (i.e.,3′) from the guide sequence or spacer sequence. In other embodiments,multiple DRs (such as dual DRs) may be present.

In certain embodiments, the crRNA comprises a stem loop, preferably asingle stem loop. In certain embodiments, the direct repeat sequenceforms a stem loop, preferably a single stem loop.

In certain embodiments, the spacer length of the guide RNA is from 15 to35 nt. In certain embodiments, the spacer length of the guide RNA is atleast 15 nucleotides. In certain embodiments, the spacer length is from15 to 17 nt, e.g., 15, 16, or 17 nt, from 17 to 20 nt, e.g., 17, 18, 19,or 20 nt, from 20 to 24 nt, e.g., 20, 21, 22, 23, or 24 nt, from 23 to25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt, e.g., 24, 25, 26, or 27nt, from 27-30 nt, e.g., 27, 28, 29, or 30 nt, from 30-35 nt, e.g., 30,31, 32, 33, 34, or 35 nt, or 35 nt or longer.

Interfering RNA (RNAi) and microRNA (miRNA)

In other embodiments, the target RNA may include interfering RNA, i.e.RNA involved in an RNA interference pathway, such as shRNA, siRNA and soforth, both in eukaryotes and prokaryotes. In other embodiments, thetarget RNA may include microRNA (miRNA). Control over interfering RNA ormiRNA may help reduce off-target effects (OTE) seen with thoseapproaches by reducing the longevity of the interfering RNA or miRNA invivo or in vitro.

In certain embodiments, the target is not the miRNA itself, but themiRNA binding site of a miRNA target.

In certain embodiments, miRNAs may be sequestered (such as includingsubcellularly relocated). In certain embodiments, miRNAs may be cut,such as without limitation at hairpins.

In certain embodiments, miRNA processing (such as including turnover) isincreased or decreased.

If the effector protein and suitable guide are selectively expressed(for example spatially or temporally under the control of a suitablepromoter, for example a tissue- or cell cycle-specific promoter and/orenhancer) then this could be used to ‘protect’ the cells or systems (invivo or in vitro) from RNAi in those cells. This may be useful inneighbouring tissues or cells where RNAi is not required or for thepurposes of comparison of the cells or tissues where the effectorprotein and suitable guide are and are not expressed (i.e. where theRNAi is not controlled and where it is, respectively). The effectorprotein may be used to control or bind to molecules comprising orconsisting of RNA, such as ribozymes, ribosomes or riboswitches. Inembodiments of the invention, the RNA guide can recruit the effectorprotein to these molecules so that the effector protein is able to bindto them.

The protein system of the invention can be applied in areas of RNAitechnologies, without undue experimentation, from this disclosure,including therapeutic, assay and other applications (see, e.g., Guidi etal., PLoS Negl Trop Dis 9(5): e0003801. doi:10.1371/journal.pntd; Crottyet al., In vivo RNAi screens: concepts and applications. Shane Crotty .. . 2015 Elsevier Ltd. Published by Elsevier Inc., PesticideBiochemistry and Physiology (Impact Factor: 2.01). 01/2015; 120. DOI:10.1016/j.pestbp.2015.01.002 and Makkonen et al., Viruses 2015, 7(4),2099-2125; doi:10.3390/v7042099), because the present applicationprovides the foundation for informed engineering of the system.

Ribosomal RNA (rRNA)

For example, azalide antibiotics such as azithromycin, are well known.They target and disrupt the 505 ribosomal subunit. The present effectorprotein, together with a suitable guide RNA to target the 505 ribosomalsubunit, may be, in some embodiments, recruited to and bind to the 505ribosomal subunit. Thus, the present effector protein in concert with asuitable guide directed at a ribosomal (especially the 50s ribosomalsubunit) target is provided. Use of this use effector protein in concertwith the suitable guide directed at the ribosomal (especially the 50sribosomal subunit) target may include antibiotic use. In particular, theantibiotic use is analogous to the action of azalide antibiotics, suchas azithromycin. In some embodiments, prokaryotic ribosomal subunits,such as the 70S subunit in prokaryotes, the 505 subunit mentioned above,the 30S subunit, as well as the 16S and 5S subunits may be targeted. Inother embodiments, eukaryotic ribosomal subunits, such as the 80Ssubunit in eukaryotes, the 60S subunit, the 40S subunit, as well as the28S, 18S. 5.8S and 5S subunits may be targeted.

In some embodiments, the effector protein may be a RNA-binding protein,optionally functionalized, as described herein. In some embodiments, theeffector protein may be a RNA-binding protein that cleaves a singlestrand of RNA. In either case, but particularly where the RNA-bindingprotein cleaves a single strand of RNA, then ribosomal function may bemodulated and, in particular, reduced or destroyed. This may apply toany ribosomal RNA and any ribosomal subunit and the sequences of rRNAare well known.

Control of ribosomal activity is thus envisaged through use of thepresent effector protein in concert with a suitable guide to theribosomal target. This may be through cleavage of, or binding to, theribosome. In particular, reduction of ribosomal activity is envisaged.This may be useful in assaying ribosomal function in vivo or in vitro,but also as a means of controlling therapies based on ribosomalactivity, in vivo or in vitro. Furthermore, control (i.e. reduction) ofprotein synthesis in an in vivo or in vitro system is envisaged, suchcontrol including antibiotic and research and diagnostic use.

Riboswitches

A riboswitch (also known as an aptozyme) is a regulatory segment of amessenger RNA molecule that binds a small molecule. This typicallyresults in a change in production of the proteins encoded by the mRNA.Thus, control of riboswitch activity is thus envisaged through use ofthe present effector protein in concert with a suitable guide to theriboswitch target. This may be through cleavage of, or binding to, theriboswitch. In particular, reduction of riboswitch activity isenvisaged. This may be useful in assaying riboswitch function in vivo orin vitro, but also as a means of controlling therapies based onriboswitch activity, in vivo or in vitro. Furthermore, control (i.e.reduction) of protein synthesis in an in vivo or in vitro system isenvisaged. This control, as for rRNA may include antibiotic and researchand diagnostic use.

Ribozymes

Ribozymes are RNA molecules having catalytic properties, analogous toenzymes (which are of course proteins). As ribozymes, both naturallyoccurring and engineered, comprise or consist of RNA, they may also betargeted by the present RNA-binding effector protein. In someembodiments, the effector protein may be a RNA-binding protein cleavesthe ribozyme to thereby disable it. Control of ribozymal activity isthus envisaged through use of the present effector protein in concertwith a suitable guide to the ribozymal target. This may be throughcleavage of, or binding to, the ribozyme. In particular, reduction ofribozymal activity is envisaged. This may be useful in assayingribozymal function in vivo or in vitro, but also as a means ofcontrolling therapies based on ribozymal activity, in vivo or in vitro.

Gene Expression, Including RNA Processing

The effector protein may also be used, together with a suitable guide,to target gene expression, including via control of RNA processing. Thecontrol of RNA processing may include RNA processing reactions such asRNA splicing, including alternative splicing, via targeting of RNApol;viral replication (in particular of satellite viruses, bacteriophagesand retroviruses, such as HBV, HBC and HIV and others listed herein)including virioids in plants; and tRNA biosynthesis. The effectorprotein and suitable guide may also be used to control RNAactivation(RNAa). RNAa leads to the promotion of gene expression, so control ofgene expression may be achieved that way through disruption or reductionof RNAa and thus less promotion of gene expression. This is discussedmore in detail below.

RNAi Screens

Identifying gene products whose knockdown is associated with phenotypicchanges, biological pathways can be interrogated and the constituentparts identified, via RNAi screens. Control may also be exerted over orduring these screens by use of the effector protein and suitable guideto remove or reduce the activity of the RNAi in the screen and thusreinstate the activity of the (previously interfered with) gene product(by removing or reducing the interference/repression).

Satellite RNAs (satRNAs) and satellite viruses may also be treated.

Control herein with reference to RNase activity generally meansreduction, negative disruption or known-down or knock out.

In Vivo RNA Applications Inhibition of Gene Expression

The target-specific RNAses provided herein allow for very specificcutting of a target RNA. The interference at RNA level allows formodulation both spatially and temporally and in a non-invasive way, asthe genome is not modified.

A number of diseases have been demonstrated to be treatable by mRNAtargeting. While most of these studies relate to administration ofsiRNA, it is clear that the RNA targeting effector proteins providedherein can be applied in a similar way.

Examples of mRNA targets (and corresponding disease treatments) areVEGF, VEGF-R1 and RTP801 (in the treatment of AMD and/or DME), Caspase 2(in the treatment of Naion) ADRB2 (in the treatment of intraocularpressure), TRPVI (in the treatment of Dry eye syndrome, Syk kinase (inthe treatment of asthma), Apo B (in the treatment ofhypercholesterolemia or hypobetalipoproteinemia), PLK1, KSP and VEGF (inthe treatment of solid tumors), Ber-Abl (in the treatment ofCML)(Burnett and Rossi Chem Biol. 2012, 19(1): 60-71)). Similarly, RNAtargeting has been demonstrated to be effective in the treatment ofRNA-virus mediated diseases such as HIV (targeting of HIV Tet and Rev),RSV (targeting of RSV nucleocapsid) and HCV (targeting of miR-122)(Burnett and Rossi Chem Biol. 2012, 19(1): 60-71).

It is further envisaged that the RNA targeting effector protein of theinvention can be used for mutation specific or allele specificknockdown. Guide RNA's can be designed that specifically target asequence in the transcribed mRNA comprising a mutation or anallele-specific sequence. Such specific knockdown is particularlysuitable for therapeutic applications relating to disorders associatedwith mutated or allele-specific gene products. For example, most casesof familial hypobetalipoproteinemia (FHBL) are caused by mutations inthe ApoB gene. This gene encodes two versions of the apolipoprotein Bprotein: a short version (ApoB-48) and a longer version (ApoB-100).Several ApoB gene mutations that lead to FHBL cause both versions ofApoB to be abnormally short. Specifically targeting and knockdown ofmutated ApoB mRNA transcripts with an RNA targeting effector protein ofthe invention may be beneficial in treatment of FHBL. As anotherexample, Huntington's disease (HD) is caused by an expansion of CAGtriplet repeats in the gene coding for the Huntingtin protein, whichresults in an abnormal protein. Specifically targeting and knockdown ofmutated or allele-specific mRNA transcripts encoding the Huntingtinprotein with an RNA targeting effector protein of the invention may bebeneficial in treatment of HD.

It is noted that in this context, and more generally fort he variousapplications as described herein, the use of a split version of the RNAtargeting effector protein can be envisaged. Indeed, this may not onlyallow increased specificity but may also be advantageous for delivery.The Cas13b is split in the sense that the two parts of the Cas13b enzymesubstantially comprise a functioning Cas13b. Ideally, the split shouldalways be so that the catalytic domain(s) are unaffected. That Cas13bmay function as a nuclease or it may be a dead-Cas13b which isessentially an RNA-binding protein with very little or no catalyticactivity, due to typically mutation(s) in its catalytic domains.

Each half of the split Cas13b may be fused to a dimerization partner. Bymeans of example, and without limitation, employing rapamycin sensitivedimerization domains, allows to generate a chemically inducible splitCas13b for temporal control of Cas13b activity. Cas13b can thus berendered chemically inducible by being split into two fragments and thatrapamycin-sensitive dimerization domains may be used for controlledreassembly of the Cas13b. The two parts of the split Cas13b can bethought of as the N′ terminal part and the C′ terminal part of the splitCas13b. The fusion is typically at the split point of the Cas13b. Inother words, the C′ terminal of the N′ terminal part of the split Cas13bis fused to one of the dimer halves, whilst the N′ terminal of the C′terminal part is fused to the other dimer half.

The Cas13b does not have to be split in the sense that the break isnewly created. The split point is typically designed in silico andcloned into the constructs. Together, the two parts of the split Cas13b,the N′ terminal and C′ terminal parts, form a full Cas13b, comprisingpreferably at least 70% or more of the wildtype amino acids (ornucleotides encoding them), preferably at least 80% or more, preferablyat least 90% or more, preferably at least 95% or more, and mostpreferably at least 99% or more of the wildtype amino acids (ornucleotides encoding them). Some trimming may be possible, and mutantsare envisaged. Non-functional domains may be removed entirely. What isimportant is that the two parts may be brought together and that thedesired Cas13b function is restored or reconstituted. The dimer may be ahomodimer or a heterodimer.

In certain embodiments, the Cas13b effector as described herein may beused for mutation-specific, or allele-specific targeting, such as formutation-specific, or allele-specific knockdown.

The RNA targeting effector protein can moreover be fused to anotherfunctional RNAse domain, such as a non-specific RNase or Argonaute 2,which acts in synergy to increase the RNAse activity or to ensurefurther degradation of the message.

Modulation of Gene Expression Through Modulation of RNA Function

Apart from a direct effect on gene expression through cleavage of themRNA, RNA targeting can also be used to impact specific aspects of theRNA processing within the cell, which may allow a more subtle modulationof gene expression. Generally, modulation can for instance be mediatedby interfering with binding of proteins to the RNA, such as for instanceblocking binding of proteins, or recruiting RNA binding proteins.Indeed, modulations can be ensured at different levels such as splicing,transport, localization, translation and turnover of the mRNA. Similarlyin the context of therapy, it can be envisaged to address (pathogenic)malfunctioning at each of these levels by using RNA-specific targetingmolecules. In these embodiments it is in many cases preferred that theRNA targeting protein is a “dead” Cas13b that has lost the ability tocut the RNA target but maintains its ability to bind thereto, such asthe mutated forms of Cas13b described herein.

A) Alternative Splicing

Many of the human genes express multiple mRNAs as a result ofalternative splicing. Different diseases have been shown to be linked toaberrant splicing leading to loss of function or gain of function of theexpressed gene. While some of these diseases are caused by mutationsthat cause splicing defects, a number of these are not. One therapeuticoption is to target the splicing mechanism directly. The RNA targetingeffector proteins described herein can for instance be used to block orpromote slicing, include or exclude exons and influence the expressionof specific isoforms and/or stimulate the expression of alternativeprotein products. Such applications are described in more detail below.

A RNA targeting effector protein binding to a target RNA can stericallyblock access of splicing factors to the RNA sequence. The RNA targetingeffector protein targeted to a splice site may block splicing at thesite, optionally redirecting splicing to an adjacent site. For instancea RNA targeting effector protein binding to the 5′ splice site bindingcan block the recruitment of the U1 component of the spliceosome,favoring the skipping of that exon. Alternatively, a RNA targetingeffector protein targeted to a splicing enhancer or silencer can preventbinding of transacting regulatory splicing factors at the target siteand effectively block or promote splicing. Exon exclusion can further beachieved by recruitment of ILF2/3 to precursor mRNA near an exon by anRNA targeting effector protein as described herein. As yet anotherexample, a glycine rich domain can be attached for recruitment of hnRNPA1 and exon exclusion (Del Gatto-Konczak et al. Mol Cell Biol. 1999January; 19(1):251-60).

In certain embodiments, through appropriate selection of gRNA, specificsplice variants may be targeted, while other splice variants will not betargeted.

In some cases the RNA targeting effector protein can be used to promoteslicing (e.g. where splicing is defective). For instance a RNA targetingeffector protein can be associated with an effector capable ofstabilizing a splicing regulatory stem-loop in order to furthersplicing. The RNA targeting effector protein can be linked to aconsensus binding site sequence for a specific splicing factor in orderto recruit the protein to the target DNA or RNA.

Examples of diseases which have been associated with aberrant splicinginclude, but are not limited to Paraneoplastic Opsoclonus MyoclonusAtaxia (or POMA), resulting from a loss of Nova proteins which regulatesplicing of proteins that function in the synapse, and Cystic Fibrosis,which is caused by defective splicing of a cystic fibrosis transmembraneconductance regulator, resulting in the production of nonfunctionalchloride channels. In other diseases aberrant RNA splicing results ingain-of-function. This is the case for instance in myotonic dystrophywhich is caused by a CUG triplet-repeat expansion (from 50 to >1500repeats) in the 3′UTR of an mRNA, causing splicing defects.

The RNA targeting effector protein can be used to include an exon byrecruiting a splicing factor (such as U1) to a 5′splicing site topromote excision of introns around a desired exon. Such recruitmentcould be mediated trough a fusion with an arginine/serine rich domain,which functions as splicing activator (Gravely B R and Maniatis T, MolCell. 1998 (5):765-71).

It is envisaged that the RNA targeting effector protein can be used toblock the splicing machinery at a desired locus, resulting in preventingexon recognition and the expression of a different protein product. Anexample of a disorder that may treated is Duchenne muscular dystrophy(DMD), which is caused by mutations in the gene encoding for thedystrophin protein. Almost all DMD mutations lead to frameshifts,resulting in impaired dystrophin translation. The RNA targeting effectorprotein can be paired with splice junctions or exonic splicing enhancers(ESEs) thereby preventing exon recognition, resulting in the translationof a partially functional protein. This converts the lethal Duchennephenotype into the less severe Becker phenotype.

B) RNA Modification

RNA editing is a natural process whereby the diversity of gene productsof a given sequence is increased by minor modification in the RNA.Typically, the modification involves the conversion of adenosine (A) toinosine (I), resulting in an RNA sequence which is different from thatencoded by the genome. RNA modification is generally ensured by the ADARenzyme, whereby the pre-RNA target forms an imperfect duplex RNA bybase-pairing between the exon that contains the adenosine to be editedand an intronic non-coding element. A classic example of A-I editing isthe glutamate receptor GluR-B mRNA, whereby the change results inmodified conductance properties of the channel (Higuchi M, et al. Cell.1993; 75:1361-70).

According to the invention, enzymatic approaches are used to inducetransitions (A<->G or C<->U changes) or transversions (any purine to anypyrimidine of vice versa) in the RNA bases of a given transcript.Transitions can be directly induced by using adenosine (ADAR1/2)) orcytosine deaminases (APOBEC, AID) which convert A to I or C to U,respectively. Transversions can be indirectly induced by localizingreactive oxygen species damage to the bases of interest, which causeschemical modifications to be added to the affected bases, such as theconversion of guanine to oxo-guanine. An oxo-gaunine is recognized as aT and will thus base pair with an adenine causing translation to beaffected. Proteins that can be recruited for ROS-mediated base damageinclude APEX and mini-SOG. With both approaches, these effectors can befused to a catalytically inactive Cas13b and be recruited to sites ontranscripts where these types of mutations are desired.

In humans, a heterozygous functional-null mutation in the ADAR1 geneleads to a skin disease, human pigmentary genodermatosis (Miyamura Y, etal. Am J Hum Genet. 2003; 73:693-9). It is envisaged that the RNAtargeting effector proteins of the present invention can be used tocorrect malfunctioning RNA modification.

It is further envisaged that RNA adenosine methylase(N(6)-methyladenosine) can be fused to the RNA targeting effectorproteins of the invention and targeted to a transcript of interest. Thismethylase causes reversible methylation, has regulatory roles and mayaffect gene expression and cell fate decisions by modulating multipleRNA-related cellular pathways (Fu et al Nat Rev Genet. 2014;15(5):293-306).

C) Polyadenylation

Polyadenylation of an mRNA is important for nuclear transport,translation efficiency and stability of the mRNA, and all of these, aswell as the process of polyadenylation, depend on specific RBPs. Mosteukaryotic mRNAs receive a 3′ poly(A) tail of about 200 nucleotidesafter transcription. Polyadenylation involves different RNA-bindingprotein complexes which stimulate the activity of a poly(A)polymerase(Minvielle-Sebastia L et al. Curr Opin Cell Biol. 1999; 11:352-7). It isenvisaged that the RNA-targeting effector proteins provided herein canbe used to interfere with or promote the interaction between theRNA-binding proteins and RNA.

Examples of diseases which have been linked to defective proteinsinvolved in polyadenylation are oculopharyngeal muscular dystrophy(OPMD) (Brais B, et al. Nat Genet. 1998; 18:164-7).

D) RNA Export

After pre-mRNA processing, the mRNA is exported from the nucleus to thecytoplasm. This is ensured by a cellular mechanism which involves thegeneration of a carrier complex, which is then translocated through thenuclear pore and releases the mRNA in the cytoplasm, with subsequentrecycling of the carrier.

Overexpression of proteins (such as TAP) which play a role in the exportof RNA has been found to increase export of transcripts that areotherwise ineffeciently exported in Xenopus (Katahira J, et al. EMBO J.1999; 18:2593-609).

E) mRNA Localization

mRNA localization ensures spatially regulated protein production.Localization of transcripts to a specific region of the cell can beensured by localization elements. In particular embodiments, it isenvisaged that the effector proteins described herein can be used totarget localization elements to the RNA of interest. The effectorproteins can be designed to bind the target transcript and shuttle themto a location in the cell determined by its peptide signal tag. Moreparticularly for instance, a RNA targeting effector protein fused to oneor more nuclear localization signal (NLS) and/or one or more nuclearexport signal (NES) can be used to alter RNA localization.

Further examples of localization signals include the zipcode bindingprotein (ZBP1) which ensures localization of β-actin to the cytoplasm inseveral asymmetric cell types, KDEL retention sequence (localization toendoplasmic reticulum), nuclear export signal (localization tocytoplasm), mitochondrial targeting signal (localization tomitochondria), peroxisomal targeting signal (localization to peroxisome)and m6A marking/YTHDF2 (localization to p-bodies). Other approaches thatare envisaged are fusion of the RNA targeting effector protein withproteins of known localization (for instance membrane, synapse).

Alternatively, the effector protein according to the invention may forinstance be used in localization-dependent knockdown. By fusing theeffector protein to a appropriate localization signal, the effector istargeted to a particular cellular compartment. Only target RNAs residingin this compartment will effectively be targeted, whereas otherwiseidentical targets, but residing in a different cellular compartment willnot be targeted, such that a localization dependent knockdown can beestablished.

F) Translation

The RNA targeting effector proteins described herein can be used toenhance or repress translation. It is envisaged that upregulatingtranslation is a very robust way to control cellular circuits. Further,for functional studies a protein translation screen can be favorableover transcriptional upregulation screens, which have the shortcomingthat upregulation of transcript does not translate into increasedprotein production.

It is envisaged that the RNA targeting effector proteins describedherein can be used to bring translation initiation factors, such asEIF4G in the vicinity of the 5′ untranslated repeat (5′UTR) of amessenger RNA of interest to drive translation (as described in DeGregorio et al. EMBO J. 1999; 18(17):4865-74 for a non-reprogrammableRNA binding protein). As another example GLD2, a cytoplasmic poly(A)polymerase, can be recruited to the target mRNA by an RNA targetingeffector protein. This would allow for directed polyadenylation of thetarget mRNA thereby stimulating translation.

Similarly, the RNA targeting effector proteins envisaged herein can beused to block translational repressors of mRNA, such as ZBP1(Huttelmaier S, et al. Nature. 2005; 438:512-5). By binding totranslation initiation site of a target RNA, translation can be directlyaffected.

In addition, fusing the RNA targeting effector proteins to a proteinthat stabilizes mRNAs, e.g. by preventing degradation thereof such asRNase inhibitors, it is possible to increase protein production from thetranscripts of interest.

It is envisaged that the RNA targeting effector proteins describedherein can be used to repress translation by binding in the 5UTR regionsof a RNA transcript and preventing the ribosome from forming andbeginning translation.

Further, the RNA targeting effector protein can be used to recruit Caf1,a component of the CCR4-NOT deadenylase complex, to the target mRNA,resulting in deadenylation or the target transcript and inhibition ofprotein translation.

For instance, the RNA targeting effector protein of the invention can beused to increase or decrease translation of therapeutically relevantproteins. Examples of therapeutic applications wherein the RNA targetingeffector protein can be used to downregulate or upregulate translationare in amyotrophic lateral sclerosis (ALS) and cardiovascular disorders.Reduced levels of the glial glutamate transporter EAAT2 have beenreported in ALS motor cortex and spinal cord, as well as multipleabnormal EAAT2 mRNA transcripts in ALS brain tissue. Loss of the EAAT2protein and function thought to be the main cause of excitotoxicity inALS. Restoration of EAAT2 protein levels and function may providetherapeutic benefit. Hence, the RNA targeting effector protein can bebeneficially used to upregulate the expression of EAAT2 protein, e.g. byblocking translational repressors or stabilizing mRNA as describedabove. Apolipoprotein A1 is the major protein component of high densitylipoprotein (HDL) and ApoA1 and HDL are generally considered asatheroprotective. It is envisages that the RNA targeting effectorprotein can be beneficially used to upregulate the expression of ApoA1,e.g. by blocking translational repressors or stabilizing mRNA asdescribed above.

G) mRNA Turnover

Translation is tightly coupled to mRNA turnover and regulated mRNAstability. Specific proteins have been described to be involved in thestability of transcripts (such as the ELAV/Hu proteins in neurons, KeeneJ D, 1999, Proc Natl Acad Sci USA. 96:5-7) and tristetraprolin (TTP).These proteins stabilize target mRNAs by protecting the messages fromdegradation in the cytoplasm (Peng S S et al., 1988, EMBO J.17:3461-70).

It can be envisaged that the RNA-targeting effector proteins of thepresent invention can be used to interfere with or to promote theactivity of proteins acting to stabilize mRNA transcripts, such thatmRNA turnover is affected. For instance, recruitment of human TTP to thetarget RNA using the RNA targeting effector protein would allow foradenylate-uridylate-rich element (AU-rich element) mediatedtranslational repression and target degradation. AU-rich elements arefound in the 3′ UTR of many mRNAs that code for proto-oncogenes, nucleartranscription factors, and cytokines and promote RNA stability. Asanother example, the RNA targeting effector protein can be fused to HuR,another mRNA stabilization protein (Hinman M N and Lou H, Cell Mol LifeSci 2008; 65:3168-81), and recruit it to a target transcript to prolongits lifetime or stabilize short-lived mRNA.

It is further envisaged that the RNA-targeting effector proteinsdescribed herein can be used to promote degradation of targettranscripts. For instance, m6A methyltransferase can be recruited to thetarget transcript to localize the transcript to P-bodies leading todegradation of the target.

As yet another example, an RNA targeting effector protein as describedherein can be fused to the non-specific endonuclease domain PilTN-terminus (PIN), to recruit it to a target transcript and allowdegradation thereof.

Patients with paraneoplastic neurological disorder (PND)-associatedencephalomyelitis and neuropathy are patients who develop autoantibodiesagainst Hu-proteins in tumors outside of the central nervous system(Szabo A et al. 1991, Cell; 67:325-33 which then cross the blood-brainbarrier. It can be envisaged that the RNA-targeting effector proteins ofthe present invention can be used to interfere with the binding ofauto-antibodies to mRNA transcripts.

Patients with dystrophy type 1 (DM1), caused by the expansion of (CUG)nin the 3′ UTR of dystrophia myotonica-protein kinase (DMPK) gene, arecharacterized by the accumulation of such transcripts in the nucleus. Itis envisaged that the RNA targeting effector proteins of the inventionfused with an endonuclease targeted to the (CUG)n repeats could inhibitsuch accumulation of aberrant transcripts.

H) Interaction with Multi-Functional Proteins

Some RNA-binding proteins bind to multiple sites on numerous RNAs tofunction in diverse processes. For instance, the hnRNP A1 protein hasbeen found to bind exonic splicing silencer sequences, antagonizing thesplicing factors, associate with telomere ends (thereby stimulatingtelomere activity) and bind miRNA to facilitate Drosha-mediatedprocessing thereby affecting maturation. It is envisaged that theRNA-binding effector proteins of the present invention can interferewith the binding of RNA-binding proteins at one or more locations.

I) RNA Folding

RNA adopts a defined structure in order to perform its biologicalactivities. Transitions in conformation among alternative tertiarystructures are critical to most RNA-mediated processes. However, RNAfolding can be associated with several problems. For instance, RNA mayhave a tendency to fold into, and be upheld in, improper alternativeconformations and/or the correct tertiary structure may not besufficiently thermodynamically favored over alternative structures. TheRNA targeting effector protein, in particular a cleavage-deficient ordead RNA targeting protein, of the invention may be used to directfolding of (m)RNA and/or capture the correct tertiary structure thereof.Use of RNA-targeting effector protein in modulating cellular status

In certain embodiments Cas13b in a complex with crRNA is activated uponbinding to target RNA and subsequently cleaves any nearby ssRNA targets(i.e. “collateral” or “bystander” effects). Cas13b, once primed by thecognate target, can cleave other (non-complementary) RNA molecules. Suchpromiscuous RNA cleavage could potentially cause cellular toxicity, orotherwise affect cellular physiology or cell status.

Accordingly, in certain embodiments, the non-naturally occurring orengineered composition, vector system, or delivery systems as describedherein are used for or are for use in induction of cell dormancy. Incertain embodiments, the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as described herein areused for or are for use in induction of cell cycle arrest. In certainembodiments, the non-naturally occurring or engineered composition,vector system, or delivery systems as derscribed herein are used for orare for use in reduction of cell growth and/or cell proliferation, Incertain embodiments, the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as derscribed herein areused for or are for use in induction of cell anergy. In certainembodiments, the non-naturally occurring or engineered composition,vector system, or delivery systems as derscribed herein are used for orare for use in induction of cell apoptosis. In certain embodiments, thenon-naturally occurring or engineered composition, vector system, ordelivery systems as derscribed herein are used for or are for use inincuction of cell necrosis. In certain embodiments, the non-naturallyoccurring or engineered composition, vector system, or delivery systemsas derscribed herein are used for or are for use in induction of celldeath. In certain embodiments, the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as described herein areused for or are for use in induction of programmed cell death.

In certain embodiments, the invention relates to a method for inductionof cell dormancy comprising introducing or inducing the non-naturallyoccurring or engineered composition, vector system, or delivery systemsas described herein. In certain embodiments, the invention relates to amethod for induction of cell cycle arrest comprising introducing orinducing the non-naturally occurring or engineered composition, vectorsystem, or delivery systems as described herein. In certain embodiments,the invention relates to a method for reduction of cell growth and/orcell proliferation comprising introducing or inducing the non-naturallyoccurring or engineered composition, vector system, or delivery systemsas derscribed herein. In certain embodiments, the invention relates to amethod for induction of cell anergy comprising introducing or inducingthe non-naturally occurring or engineered composition, vector system, ordelivery systems as derscribed herein. In certain embodiments, theinvention relates to a method for induction of cell apoptosis comprisingintroducing or inducing the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as derscribed herein. Incertain embodiments, the invention relates to a method for induction ofcell necrosis comprising introducing or inducing the non-naturallyoccurring or engineered composition, vector system, or delivery systemsas derscribed herein. In certain embodiments, the invention relates to amethod for induction of cell death comprising introducing or inducingthe non-naturally occurring or engineered composition, vector system, ordelivery systems as described herein. In certain embodiments, theinvention relates to a method for induction of programmed cell deathcomprising introducing or inducing the non-naturally occurring orengineered composition, vector system, or delivery systems as describedherein.

The methods and uses as described herein may be therapeutic orprophylactic and may target particular cells, cell (sub)populations, orcell/tissue types. In particular, the methods and uses as describedherein may be therapeutic or prophylactic and may target particularcells, cell (sub)populations, or cell/tissue types expressing one ormore target sequences, such as one or more particular target RNA (e.g.ss RNA). Without limitation, target cells may for instance be cancercells expressing a particular transcript, e.g. neurons of a given class,(immune) cells causing e.g. autoimmunity, or cells infected by aspecific (e.g. viral) pathogen, etc.

Accordingly, in certain embodiments, the invention relates to a methodfor treating a pathological condition characterized by the presence ofundersirable cells (host cells), comprising introducing or inducing thenon-naturally occurring or engineered composition, vector system, ordelivery systems as derscribed herein. In certain embodiments, theinvention relates the use of the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as derscribed herein fortreating a pathological condition characterized by the presence ofundersirable cells (host cells). In certain embodiments, the inventionrelates the non-naturally occurring or engineered composition, vectorsystem, or delivery systems as derscribed herein for use in treating apathological condition characterized by the presence of undesirablecells (host cells). It is to be understood that preferably theCRISPR-Cas system targets a target specific for the undesirable cells.In certain embodiments, the invention relates to the use of thenon-naturally occurring or engineered composition, vector system, ordelivery systems as described herein for treating, preventing, oralleviating cancer. In certain embodiments, the invention relates to thenon-naturally occurring or engineered composition, vector system, ordelivery systems as described herein for use in treating, preventing, oralleviating cancer. In certain embodiments, the invention relates to amethod for treating, preventing, or alleviating cancer comprisingintroducing or inducing the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as derscribed herein. Itis to be understood that preferably the CRISPR-Cas system targets atarget specific for the cancer cells. In certain embodiments, theinvention relates to the use of the non-naturally occurring orengineered composition, vector system, or delivery systems as derscribedherein for treating, preventing, or alleviating infection of cells by apathogen. In certain embodiments, the invention relates to thenon-naturally occurring or engineered composition, vector system, ordelivery systems as derscribed herein for use in treating, preventing,or alleviating infection of cells by a pathogen. In certain embodiments,the invention relates to a method for treating, preventing, oralleviating infection of cells by a pathogen comprising introducing orinducing the non-naturally occurring or engineered composition, vectorsystem, or delivery systems as derscribed herein. It is to be understoodthat preferably the CRISPR-Cas system targets a target specific for thecells infected by the pathogen (e.g. a pathogen derived target). Incertain embodiments, the invention relates to the use of thenon-naturally occurring or engineered composition, vector system, ordelivery systems as described herein for treating, preventing, oralleviating an autoimmune disorder. In certain embodiments, theinvention relates to the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as described herein foruse in treating, preventing, or alleviating an autoimmune disorder. Incertain embodiments, the invention relates to a method for treating,preventing, or alleviating an autoimmune disorder comprising introducingor inducing the non-naturally occurring or engineered composition,vector system, or delivery systems as described herein. It is to beunderstood that preferably the CRISPR-Cas system targets a targetspecific for the cells responsible for the autoimmune disorder (e.g.specific immune cells).

Use of RNA-Targeting Effector Protein in RNA Detection or ProteinDetection

It is further envisaged that the RNA targeting effector protein can beused for detection of nucleic acids or proteins in a biological sample.The samples can be can be cellular or cell-free.

It is further envisaged that the RNA targeting effector protein can beused in Northern blot assays. Northern blotting involves the use ofelectrophoresis to separate RNA samples by size. The RNA targetingeffector protein can be used to specifically bind and detect the targetRNA sequence.

A RNA targeting effector protein can also be fused to a fluorescentprotein (such as GFP) and used to track RNA localization in livingcells. More particularly, the RNA targeting effector protein can beinactivated in that it no longer cleaves RNA. In particular embodiments,it is envisaged that a split RNA targeting effector protein can be used,whereby the signal is dependent on the binding of both subproteins, inorder to ensure a more precise visualization. Alternatively, a splitfluorescent protein can be used that is reconstituted when multiple RNAtargeting effector protein complexes bind to the target transcript. Itis further envisaged that a transcript is targeted at multiple bindingsites along the mRNA so the fluorescent signal can amplify the truesignal and allow for focal identification. As yet another alternative,the fluorescent protein can be reconstituted form a split intein.

RNA targeting effector proteins are for instance suitably used todetermine the localization of the RNA or specific splice variants, thelevel of mRNA transcript, up- or down regulation of transcripts anddisease-specific diagnosis. The RNA targeting effector proteins can beused for visualization of RNA in (living) cells using e.g. fluorescentmicroscopy or flow cytometry, such as fluorescence-activated cellsorting (FACS) which allows for high-throughput screening of cells andrecovery of living cells following cell sorting. Further, expressionlevels of different transcripts can be assessed simultaneously understress, e.g. inhibition of cancer growth using molecular inhibitors orhypoxic conditions on cells. Another application would be to tracklocalization of transcripts to synaptic connections during a neuralstimulus using two photon microscopy.

In certain embodiments, the components or complexes according to theinvention as described herein can be used in multiplexed error-robustfluorescence in situ hybridization (MERFISH; Chen et al. Science; 2015;348(6233)), such as for instance with (fluorescently) labeled Cas13beffectors.

In Vitro Apex Labeling

Cellular processes depend on a network of molecular interactions amongprotein, RNA, and DNA. Accurate detection of protein—DNA and protein—RNAinteractions is key to understanding such processes. In vitro proximitylabeling technology employs an affinity tag combined with e.g. aphotoactivatable probe to label polypeptides and RNAs in the vicinity ofa protein or RNA of interest in vitro. After UV irradiation thephotoactivatable group reacts with proteins and other molecules that arein close proximity to the tagged molecule, thereby labelling them.Labelled interacting molecules can subsequently be recovered andidentified. The RNA targeting effector protein of the invention can forinstance be used to target a probe to a selected RNA sequence.

These applications could also be applied in animal models for in vivoimaging of disease relevant applications or difficult-to culture celltypes.

The invention provides agents and methods for diagnosing and monitoringhealth states through non-invasive sampling of cell free RNA, includingtesting for risk and guiding RNA-targeted therapies, and is useful insetting where rapid administration of therapy is important to treatmentoutcomes. In one embodiment, the invention provides cancer detectionmethods and agents for circulating tumor RNA, including for monitoringrecurrence and/or development of common drug resistance mutations. Inanother embodiment, the invention provides detection methods and agentsfor detection and/or identification of bacterial species directly fromblood or serum to monitor, e.g., disease progression and sepsis. In anembodiment of the invention, the Cas13b proteins and derivatives areused to distinguish and diagnose common diseases such as rhinovirus orupper respiratory tract infections from more serious infections such asbronchitis.

The invention provides methods and agents for rapid genotyping foremergency pharmacogenomics, including guidance for administration ofanticoagulants during myocardial infarction or stroke treatment basedon, e.g., VKORC1, CYP2C9, and CYP2C19 genotyping.

The invention provides agents and methods for monitoring foodcontamination by bacteria at all points along a food production anddelivery chain. In another embodiment, the invention provides forquality control and monitoring, e.g. by identification of food sourcesand determination of purity. In one non-limiting example, the inventionmay be used to identify or confirm a food sources, such as a species ofanimal meat and seafood.

In another embodiment, the invention is used in phorensicdeterminations. For example, crime scene samples containing blood orother bodily fluids. In an embodiment of the invention, the invention isused to identify nucleic acid samples from fingerprints.

Use of RNA-Targeting Effector Protein in RNA Origami/In Vitro AssemblyLines—Combinatorics

RNA origami refers to nanoscale folded structures for creatingtwo-dimensional or three-dimensional structures using RNA as integratedtemplate. The folded structure is encoded in the RNA and the shape ofthe resulting RNA is thus determined by the synthesized RNA sequence(Geary, et al. 2014. Science, 345 (6198). pp. 799-804). The RNA origamimay act as scaffold for arranging other components, such as proteins,into complexes. The RNA targeting effector protein of the invention canfor instance be used to target proteins of interest to the RNA origamiusing a suitable guide RNA.

Use of RNA-Targeting Effector Protein in RNA Isolation or Purification,Enrichment or Depletion

It is further envisages that the RNA targeting effector protein whencomplexed to RNA can be used to isolate and/or purify the RNA. The RNAtargeting effector protein can for instance be fused to an affinity tagthat can be used to isolate and/or purify the RNA-RNA targeting effectorprotein complex. Such applications are for instance useful in theanalysis of gene expression profiles in cells. In particularembodiments, it can be envisaged that the RNA targeting effectorproteins can be used to target a specific noncoding RNA (ncRNA) therebyblocking its activity, providing a useful functional probe. In certainembodiments, the effecetor protein as described herein may be used tospecifically enrich for a particular RNA (including but not limited toincreasing stability, etc.), or alternatively to specifically deplete aparticular RNA (such as without limitation for instance particularsplice variants, isoforms, etc.).

Interrogation of lincRNA Function and Other Nuclear RNAs

Current RNA knockdown strategies such as siRNA have the disadvantagethat they are mostly limited to targeting cytosolic transcripts sincethe protein machinery is cytosolic. The advantage of a RNA targetingeffector protein of the present invention, an exogenous system that isnot essential to cell function, is that it can be used in anycompartment in the cell. By fusing a NLS signal to the RNA targetingeffector protein, it can be guided to the nucleus, allowing nuclear RNAsto be targeted. It is for instance envisaged to probe the function oflincRNAs. Long intergenic non-coding RNAs (lincRNAs) are a vastlyunderexplored area of research. Most lincRNAs have as of yet unknownfunctions which could be studies using the RNA targeting effectorprotein of the invention.

Identification of RNA Binding Proteins

Identifying proteins bound to specific RNAs can be useful forunderstanding the roles of many RNAs. For instance, many lincRNAsassociate with transcriptional and epigenetic regulators to controltranscription. Understanding what proteins bind to a given lincRNA canhelp elucidate the components in a given regulatory pathway. A RNAtargeting effector protein of the invention can be designed to recruit abiotin ligase to a specific transcript in order to label locally boundproteins with biotin. The proteins can then be pulled down and analyzedby mass spectrometry to identify them.

Assembly of Complexes on RNA and Substrate Shuttling

RNA targeting effector proteins of the invention can further be used toassemble complexes on RNA. This can be achieved by functionalizing theRNA targeting effector protein with multiple related proteins (e.g.components of a particular synthesis pathway). Alternatively, multipleRNA targeting effector proteins can be functionalized with suchdifferent related proteins and targeted to the same or adjacent targetRNA. Useful application of assembling complexes on RNA are for instancefacilitating substrate shuttling between proteins.

Synthetic Biology

The development of biological systems have a wide utility, including inclinical applications. It is envisaged that the programmable RNAtargeting effector proteins of the invention can be used fused to splitproteins of toxic domains for targeted cell death, for instance usingcancer-linked RNA as target transcript. Further, pathways involvingprotein-protein interaction can be influenced in synthetic biologicalsystems with e.g. fusion complexes with the appropriate effectors suchas kinases or other enzymes.

Protein Splicing: Inteins

Protein splicing is a post-translational process in which an interveningpolypeptide, referred to as an intein, catalyzes its own excision fromthe polypeptides Hacking it, referred to as exteins, as well assubsequent ligation of the exteins. The assembly of two or more RNAtargeting effector proteins as described herein on a target transcriptcould be used to direct the release of a split intein (Topilina andMills Mob DNA. 2014 Feb. 4; 5(1):5), thereby allowing for directcomputation of the existence of a mRNA transcript and subsequent releaseof a protein product, such as a metabolic enzyme or a transcriptionfactor (for downstream actuation of transcription pathways). Thisapplication may have significant relevance in synthetic biology (seeabove) or large-scale bioproduction (only produce product under certainconditions).

Inducible, Dosed and Self-Inactivating Systems

In one embodiment, fusion complexes comprising an RNA targeting effectorprotein of the invention and an effector component are designed to beinducible, for instance light inducible or chemically inducible. Suchinducibility allows for activation of the effector component at adesired moment in time.

Light inducibility is for instance achieved by designing a fusioncomplex wherein CRY2 PHR/CIBN pairing is used for fusion. This system isparticularly useful for light induction of protein interactions inliving cells (Konermann S, et al. Nature. 2013; 500:472-476).

Chemical inducibility is for instance provided for by designing a fusioncomplex wherein FKBP/FRB (FK506 binding protein/FKBP rapamycin binding)pairing is used for fusion. Using this system rapamycin is required forbinding of proteins (Zetsche et al. Nat Biotechnol. 2015; 33(2):139-42describes the use of this system for Cas9).

Further, when introduced in the cell as DNA, the RNA targeting effectorprotein of the inventions can be modulated by inducible promoters, suchas tetracycline or doxycycline controlled transcriptional activation(Tet-On and Tet-Off expression system), hormone inducible geneexpression system such as for instance an ecdysone inducible geneexpression system and an arabinose-inducible gene expression system.When delivered as RNA, expression of the RNA targeting effector proteincan be modulated via a riboswitch, which can sense a small molecule liketetracycline (as described in Goldfless et al. Nucleic Acids Res. 2012;40(9):e64).

In one embodiment, the delivery of the RNA targeting effector protein ofthe invention can be modulated to change the amount of protein or crRNAin the cell, thereby changing the magnitude of the desired effect or anyundesired off-target effects.

In one embodiment, the RNA targeting effector proteins described hereincan be designed to be self-inactivating. When delivered to a cell asRNA, either mRNA or as a replication RNA therapeutic (Wrobleska et alNat Biotechnol. 2015 August; 33(8): 839-841), they can self-inactivateexpression and subsequent effects by destroying the own RNA, therebyreducing residency and potential undesirable effects.

For further in vivo applications of RNA targeting effector proteins asdescribed herein, reference is made to Mackay J P et al (Nat Struct MolBiol. 2011 March; 18(3):256-61), Nelles et al (Bioessays. 2015 July;37(7):732-9) and Abil Z and Zhao H (Mol Biosyst. 2015 October;11(10):2658-65), which are incorporated herein by reference. Inparticular, the following applications are envisaged in certainembodiments of the invention, preferably in certain embodiments by usingcatalytically inactive Cas13b: enhancing translation (e.g.Cas13b—translation promotion factor fusions (e.g. eIF4 fusions));repressing translation (e.g. gRNA targeting ribosome binding sites);exon skipping (e.g. gRNAs targeting splice donor and/or acceptor sites);exon inclusion (e.g. gRNA targeting a particular exon splice donorand/or acceptor site to be included or Cas13b fused to or recruitingspliceosome components (e.g. U1 snRNA)); accessing RNA localization(e.g. Cas13b—marker fusions (e.g. EGFP fusions)); altering RNAlocalization (e.g. Cas13b—localization signal fusions (e.g. NLS or NESfusions)); RNA degradation (in this case no catalytically inactiveCas13b is to be used if relied on the activity of Cas13b, alternativelyand for increased specificity, a split Cas13b may be used); inhibitionof non-coding RNA function (e.g. miRNA), such as by degradation orbinding of gRNA to functional sites (possibly titrating out at specificsites by relocalization by Cas13b-signal sequence fusions).

Cas13b function is robust to 5′ or 3′ extensions of the crRNA and toextension of the crRNA loop. It is therefore envisages that MS2 loopsand other recruitment domains can be added to the crRNA withoutaffecting complex formation and binding to target transcripts. Suchmodifications to the crRNA for recruitment of various effector domainsare applicable in the uses of a RNA targeted effector proteins describedabove.

Cas13b is capable of mediating resistance to RNA phages. It is thereforeenvisaged that Cas13b can be used to immunize, e.g. animals, humans andplants, against RNA-only pathogens, including but not limited toretroviruses (e.g lentiviruses, such as HIV), HCV, Ebola virus and Zikavirus.

In certain embodiments, Cas13b can process (cleave) its own array. Thisapplies to both the wildtype Cas13b protein and the mutated Cas13bprotein containing one or more mutated amino acid residues asherein-discussed. It is therefore envisaged that multiple crRNAsdesigned for different target transcripts and/or applications can bedelivered as a single pre-crRNA or as a single transcript driven by onepromotor. Such method of delivery has the advantages that it issubstantially more compact, easier to synthesize and easier to deliveryin viral systems. It will be understood that exact amino acid positionsmay vary for orthologues of a herein Cas13b can be adequately determinedby protein alignment, as is known in the art, and as described hereinelsewhere. Aspects of the invention also encompass methods and uses ofthe compositions and systems described herein in genome engineering,e.g. for altering or manipulating the expression of one or more genes orthe one or more gene products, in prokaryotic or eukaryotic cells, invitro, in vivo or ex vivo.

Aspects of the invention also encompass methods and uses of thecompositions and systems described herein in genome or transcriptomeengineering, e.g. for altering or manipulating the (protein) expressionof one or more genes or the one or more gene products, in prokaryotic oreukaryotic cells, in vitro, in vivo or ex vivo.

In an aspect, the invention provides methods and compositions formodulating, e.g., reducing, (protein) expression of a target RNA incells. In the subject methods, a Cas13b system of the invention isprovided that interferes with transcription, stability, and/ortranslation of an RNA.

In certain embodiments, an effective amount of Cas13b system is used tocleave RNA or otherwise inhibit RNA expression. In this regard, thesystem has uses similar to siRNA and shRNA, thus can also be substitutedfor such methods. The method includes, without limitation, use of aCas13b system as a substitute for e.g., an interfering ribonucleic acid(such as an siRNA or shRNA) or a transcription template thereof, e.g., aDNA encoding an shRNA. The Cas13b system is introduced into a targetcell, e.g., by being administered to a mammal that includes the targetcell,

Advantageously, a Cas13b system of the invention is specific. Forexample, whereas interfering ribonucleic acid (such as an siRNA orshRNA) polynucleotide systems are plagued by design and stability issuesand off-target binding, a Cas13b system of the invention can be designedwith high specificity.

Destabilized Cas13b

In certain embodiments, the effector protein (CRISPR enzyme; Cas13b)according to the invention as described herein is associated with orfused to a destabilization domain (DD). In some embodiments, the DD isER50. A corresponding stabilizing ligand for this DD is, in someembodiments, 4HT. As such, in some embodiments, one of the at least oneDDs is ER50 and a stabilizing ligand therefor is 4HT. or CMP8 In someembodiments, the DD is DHFR50. A corresponding stabilizing ligand forthis DD is, in some embodiments, TMP. As such, in some embodiments, oneof the at least one DDs is DHFR50 and a stabilizing ligand therefor isTMP. In some embodiments, the DD is ER50. A corresponding stabilizingligand for this DD is, in some embodiments, CMP8. CMP8 may therefore bean alternative stabilizing ligand to 4HT in the ER50 system. While itmay be possible that CMP8 and 4HT can/should be used in a competitivematter, some cell types may be more susceptible to one or the other ofthese two ligands, and from this disclosure and the knowledge in the artthe skilled person can use CMP8 and/or 4HT.

In some embodiments, one or two DDs may be fused to the N-terminal endof the CRISPR enzyme with one or two DDs fused to the C-terminal of theCRISPR enzyme. In some embodiments, the at least two DDs are associatedwith the CRISPR enzyme and the DDs are the same DD, i.e. the DDs arehomologous. Thus, both (or two or more) of the DDs could be ER50 DDs.This is preferred in some embodiments. Alternatively, both (or two ormore) of the DDs could be DHFR50 DDs. This is also preferred in someembodiments. In some embodiments, the at least two DDs are associatedwith the CRISPR enzyme and the DDs are different DDs, i.e. the DDs areheterologous. Thus, one of the DDS could be ER50 while one or more ofthe DDs or any other DDs could be DHFR50. Having two or more DDs whichare heterologous may be advantageous as it would provide a greater levelof degradation control. A tandem fusion of more than one DD at the N orC-term may enhance degradation; and such a tandem fusion can be, forexample ER50-ER50-Cas13b or DHFR-DHFR-Cas13b It is envisaged that highlevels of degradation would occur in the absence of either stabilizingligand, intermediate levels of degradation would occur in the absence ofone stabilizing ligand and the presence of the other (or another)stabilizing ligand, while low levels of degradation would occur in thepresence of both (or two of more) of the stabilizing ligands. Controlmay also be imparted by having an N-terminal ER50 DD and a C-terminalDHFR50 DD.

In some embodiments, the fusion of the CRISPR enzyme with the DDcomprises a linker between the DD and the CRISPR enzyme. In someembodiments, the linker is a GlySer linker. In some embodiments, theDD-CRISPR enzyme further comprises at least one Nuclear Export Signal(NES). In some embodiments, the DD-CRISPR enzyme comprises two or moreNESs. In some embodiments, the DD-CRISPR enzyme comprises at least oneNuclear Localization Signal (NLS). This may be in addition to an NES. Insome embodiments, the CRISPR enzyme comprises or consists essentially ofor consists of a localization (nuclear import or export) signal as, oras part of, the linker between the CRISPR enzyme and the DD. HA or Flagtags are also within the ambit of the invention as linkers. Applicantsuse NLS and/or NES as linker and also use Glycine Serine linkers asshort as GS up to (GGGGS)₃.

Destabilizing domains have general utility to confer instability to awide range of proteins; see, e.g., Miyazaki, J Am Chem Soc. Mar. 7,2012; 134(9): 3942-3945, incorporated herein by reference. CMP8 or4-hydroxytamoxifen can be destabilizing domains. More generally, Atemperature-sensitive mutant of mammalian DHFR (DHFRts), a destabilizingresidue by the N-end rule, was found to be stable at a permissivetemperature but unstable at 37° C. The addition of methotrexate, ahigh-affinity ligand for mammalian DHFR, to cells expressing DHFRtsinhibited degradation of the protein partially. This was an importantdemonstration that a small molecule ligand can stabilize a proteinotherwise targeted for degradation in cells. A rapamycin derivative wasused to stabilize an unstable mutant of the FRB domain of mTOR (FRB*)and restore the function of the fused kinase, GSK-3β.6,7 This systemdemonstrated that ligand-dependent stability represented an attractivestrategy to regulate the function of a specific protein in a complexbiological environment. A system to control protein activity can involvethe DD becoming functional when the ubiquitin complementation occurs byrapamycin induced dimerization of FK506-binding protein and FKBP12.Mutants of human FKBP12 or ecDHFR protein can be engineered to bemetabolically unstable in the absence of their high-affinity ligands,Shield-1 or trimethoprim (TMP), respectively. These mutants are some ofthe possible destabilizing domains (DDs) useful in the practice of theinvention and instability of a DD as a fusion with a CRISPR enzymeconfers to the CRISPR protein degradation of the entire fusion proteinby the proteasome. Shield-1 and TMP bind to and stabilize the DD in adose-dependent manner. The estrogen receptor ligand binding domain(ERLBD, residues 305-549 of ERS1) can also be engineered as adestabilizing domain. Since the estrogen receptor signaling pathway isinvolved in a variety of diseases such as breast cancer, the pathway hasbeen widely studied and numerous agonist and antagonists of estrogenreceptor have been developed. Thus, compatible pairs of ERLBD and drugsare known. There are ligands that bind to mutant but not wild-type formsof the ERLBD. By using one of these mutant domains encoding threemutations (L384M, M421G, G521R), it is possible to regulate thestability of an ERLBD-derived DD using a ligand that does not perturbendogenous estrogen-sensitive networks. An additional mutation (Y537S)can be introduced to further destabilize the ERLBD and to configure itas a potential DD candidate. This tetra-mutant is an advantageous DDdevelopment. The mutant ERLBD can be fused to a CRISPR enzyme and itsstability can be regulated or perturbed using a ligand, whereby theCRISPR enzyme has a DD. Another DD can be a 12-kDa (107-amino-acid) tagbased on a mutated FKBP protein, stabilized by Shield1 ligand; see,e.g., Nature Methods 5, (2008). For instance a DD can be a modifiedFK506 binding protein 12 (FKBP12) that binds to and is reversiblystabilized by a synthetic, biologically inert small molecule, Shield-1;see, e.g., Banaszynski L A, Chen L C, Maynard-Smith L A, Ooi A G,Wandless T J. A rapid, reversible, and tunable method to regulateprotein function in living cells using synthetic small molecules. Cell.2006; 126:995-1004; Banaszynski L A, Sellmyer M A, Contag C H, WandlessT J, Thorne S H. Chemical control of protein stability and function inliving mice. Nat Med. 2008; 14:1123-1127; Maynard-Smith L A, Chen L C,Banaszynski L A, Ooi A G, Wandless T J. A directed approach forengineering conditional protein stability using biologically silentsmall molecules. The Journal of biological chemistry. 2007;282:24866-24872; and Rodriguez, Chem Biol. Mar. 23, 2012; 19(3):391-398—all of which are incorporated herein by reference and may beemployed in the practice of the invention in selected a DD to associatewith a CRISPR enzyme in the practice of this invention. As can be seen,the knowledge in the art includes a number of DDs, and the DD can beassociated with, e.g., fused to, advantageously with a linker, to aCRISPR enzyme, whereby the DD can be stabilized in the presence of aligand and when there is the absence thereof the DD can becomedestabilized, whereby the CRISPR enzyme is entirely destabilized, or theDD can be stabilized in the absence of a ligand and when the ligand ispresent the DD can become destabilized; the DD allows the CRISPR enzymeand hence the CRISPR-Cas complex or system to be regulated orcontrolled—turned on or off so to speak, to thereby provide means forregulation or control of the system, e.g., in an in vivo or in vitroenvironment. For instance, when a protein of interest is expressed as afusion with the DD tag, it is destabilized and rapidly degraded in thecell, e.g., by proteasomes. Thus, absence of stabilizing ligand leads toa D associated Cas being degraded. When a new DD is fused to a proteinof interest, its instability is conferred to the protein of interest,resulting in the rapid degradation of the entire fusion protein. Peakactivity for Cas is sometimes beneficial to reduce off-target effects.Thus, short bursts of high activity are preferred. The present inventionis able to provide such peaks. In some senses the system is inducible.In some other senses, the system repressed in the absence of stabilizingligand and de-repressed in the presence of stabilizing ligand.

Application of RNA targeting—CRISPR system to plants and yeast

Definitions

In general, the term “plant” relates to any various photosynthetic,eukaryotic, unicellular or multicellular organism of the kingdom Plantaecharacteristically growing by cell division, containing chloroplasts,and having cell walls comprised of cellulose. The term plant encompassesmonocotyledonous and dicotyledonous plants. Specifically, the plants areintended to comprise without limitation angiosperm and gymnosperm plantssuch as acacia, alfalfa, amaranth, apple, apricot, artichoke, ash tree,asparagus, avocado, banana, barley, beans, beet, birch, beech,blackberry, blueberry, broccoli, Brussel's sprouts, cabbage, canola,cantaloupe, carrot, cassava, cauliflower, cedar, a cereal, celery,chestnut, cherry, Chinese cabbage, citrus, clementine, clover, coffee,corn, cotton, cowpea, cucumber, cypress, eggplant, elm, endive,eucalyptus, fennel, figs, fir, geranium, grape, grapefruit, groundnuts,ground cherry, gum hemlock, hickory, kale, kiwifruit, kohlrabi, larch,lettuce, leek, lemon, lime, locust, pine, maidenhair, maize, mango,maple, melon, millet, mushroom, mustard, nuts, oak, oats, oil palm,okra, onion, orange, an ornamental plant or flower or tree, papaya,palm, parsley, parsnip, pea, peach, peanut, pear, peat, pepper,persimmon, pigeon pea, pine, pineapple, plantain, plum, pomegranate,potato, pumpkin, radicchio, radish, rapeseed, raspberry, rice, rye,sorghum, safflower, sallow, soybean, spinach, spruce, squash,strawberry, sugar beet, sugarcane, sunflower, sweet potato, sweet corn,tangerine, tea, tobacco, tomato, trees, triticale, turf grasses,turnips, vine, walnut, watercress, watermelon, wheat, yams, yew, andzucchini. The term plant also encompasses Algae, which are mainlyphotoautotrophs unified primarily by their lack of roots, leaves andother organs that characterize higher plants.

The methods for modulating gene expression using the RNA targetingsystem as described herein can be used to confer desired traits onessentially any plant. A wide variety of plants and plant cell systemsmay be engineered for the desired physiological and agronomiccharacteristics described herein using the nucleic acid constructs ofthe present disclosure and the various transformation methods mentionedabove. In preferred embodiments, target plants and plant cells forengineering include, but are not limited to, those monocotyledonous anddicotyledonous plants, such as crops including grain crops (e.g., wheat,maize, rice, millet, barley), fruit crops (e.g., tomato, apple, pear,strawberry, orange); forage crops (e.g., alfalfa), root vegetable crops(e.g., carrot, potato, sugar beets, yam), leafy vegetable crops (e.g.,lettuce, spinach); flowering plants (e.g., petunia, rose,chrysanthemum), conifers and pine trees (e.g., pine fir, spruce); plantsused in phytoremediation (e.g., heavy metal accumulating plants); oilcrops (e.g., sunflower, rape seed) and plants used for experimentalpurposes (e.g., Arabidopsis). Thus, the methods and CRISPR-Cas systemscan be used over a broad range of plants, such as for example withdicotyledonous plants belonging to the orders Magniolales, Illiciales,Laurales, Piperales, Aristochiales, Nymphaeales, Ranunculales,Papeverales, Sarraceniaceae, Trochodendrales, Hamamelidales, Eucomiales,Leitneriales, Myri cales, agales, Casuarinales, Caryophyllales, Batales,Polygonales, Plumbaginales, Dilleniales, Theales, Malvales, IJrticales,Lecythidales, Violales, Salicales, Capparales, Ericales, Diapensales,Ebenales, Primulales, Rosales, Fabales, Podostemales; Haloragales;Myrtales, Cornales, Proteales, San tales, Rafflesiales, Celastrales,Euphorbiales, Rhamnales, Sapindales, Juglandales, Geraniales,Polygalales, Umbellales, Gentianales; Polemoniales, Lamiales,Plantaginales, Scrophulariales, Campanulales, Rubiales, Dipsacales, andAsterales; the methods and CRISPR-Cas systems can be used withmonocotyledonous plants such as those belonging to the ordersAlismatales, Hydrocharitales, Najadales, Triuridales, Commelinales,Eriocaulales, Restionales, Poales, Juncales, Cyperales, Typhales,Bromeliales, Zingiberales, Arecales, Cyclanthales, Pandanales; Arales,Lilliales; and Orchid ales, or with plants belonging to Gymnospermae,e.g those belonging to the orders Pinales, Ginkgoales, Cycadales,Araucariales, Cupressales and Gnetales.

The RNA targeting CRISPR systems and methods of use described herein canbe used over a broad range of plant species, included in thenon-limitative list of dicot, monocot or gymnosperm genera hereunder:Atropa, Alseodaphne, Anacardium, Arachis, Beilschmiedia, Brassica,Carthamus, Cocculus, Croton, Cucumis, Citrus, Citrullus, Capsicum,Catharanthus, Cocos, Coffea, Cucurbita, Amens, Duguetia, Eschscholzia,Ficus, Fragaria, Glaucium, Glycine, Gossypium, Helianthus, Hevea,Hyoscyamus, Lactuca, Landolphia, Linum, Litsea, lycopersicon, Lupinus,Manihot, Majorana, latus, Medicago, Nicotiana, Olea, Parthenium,Papaver, Persea, Phaseolus, Pistacia, Pisum, Pyrus, Prunus, Raphanus,Ricinus, Senecio, Sillomenilrm, Stephania, Sinapis, Solanum, Theobroma,Trifotium, Trigonella, Vida, Vinca, Vilis, and Vigna; and the generaAllium, Andropogort, Aragrostis, Asparagus, Avena, cynodon, Elaeis,Festuca, Festulolium, Heterocallis, Hordeum, Lonna, Lotium, Musa, Oryza,Panicum, Pannesetum, Phleum, Poa, Secale, Sorghum, Triticum, Zea, Abies,Cunninghamia, Ephedra, Picea, Pima, and Pseudotsuga.

The RNA targeting CRISPR systems and methods of use can also be usedover a broad range of “algae” or “algae cells”; including for examplealgae selected from several eukaryotic phyla, including the Rhodophyta(red algae); Chlorophyta (green algae), Phaeophyta (brown algae),Bacillariophyta (diatoms), Eustigmatophyta and dinoflagellates as wellas the prokaryotic phylum Cyanobacteria (blue-green algae). The term“algae” includes for example algae selected from: Amphora, Anabaena,Anikstrodesmis, Botryococcus, Chaetoceros, Chlamydomonas, Chlorella,Chlorococcum, Cyclotella, Cylindrotheca, Dunaliella; Emiliana, Euglena,Hematococcus; Isochrysis, Monochrysis, Monoraphidium, Nannochloris,Nannnochloropsis, Navicula, Nephrochloris, Nephroselmis, Nitzschia,Nodularia, Nostoc, Oochromonas, Oocystis; Oscillartoria, Pavlova,Phaeodactylum, Playtmonas, Pleurochrysis, Porhyra, Pseudoanabaena,Pyramimonas, Stichococcus, Synechococcus, Synechocystis, Tetraselmis,Thalassiosira, and Trichodesmium.

A part of a plant, i.e., a “plant tissue” may be treated according tothe methods of the present invention to produce an improved plant. Planttissue also encompasses plant cells. The term “plant cell” as usedherein refers to individual units of a living plant, either in an intactwhole plant or in an isolated form grown in in vitro tissue cultures, onmedia or agar, in suspension in a growth media or buffer or as a part ofhigher organized unites, such as, for example, plant tissue, a plantorgan, or a whole plant.

A “protoplast” refers to a plant cell that has had its protective cellwall completely or partially removed using, for example, mechanical orenzymatic means resulting in an intact biochemical competent unit ofliving plant that can reform their cell wall, proliferate and regenerategrow into a whole plant under proper growing conditions.

The term “transformation” broadly refers to the process by which a planthost is genetically modified by the introduction of DNA by means ofAgrobacteria or one of a variety of chemical or physical methods. Asused herein, the term “plant host” refers to plants, including anycells, tissues, organs, or progeny of the plants. Many suitable planttissues or plant cells can be transformed and include, but are notlimited to, protoplasts, somatic embryos, pollen, leaves, seedlings,stems, calli, stolons, microtubers, and shoots. A plant tissue alsorefers to any clone of such a plant, seed, progeny, propagule whethergenerated sexually or asexually, and descendents of any of these, suchas cuttings or seed.

The term “transformed” as used herein, refers to a cell, tissue, organ,or organism into which a foreign DNA molecule, such as a construct, hasbeen introduced. The introduced DNA molecule may be integrated into thegenomic DNA of the recipient cell, tissue, organ, or organism such thatthe introduced DNA molecule is transmitted to the subsequent progeny. Inthese embodiments, the “transformed” or “transgenic” cell or plant mayalso include progeny of the cell or plant and progeny produced from abreeding program employing such a transformed plant as a parent in across and exhibiting an altered phenotype resulting from the presence ofthe introduced DNA molecule. Preferably, the transgenic plant is fertileand capable of transmitting the introduced DNA to progeny through sexualreproduction.

The term “progeny”, such as the progeny of a transgenic plant, is onethat is born of, begotten by, or derived from a plant or the transgenicplant. The introduced DNA molecule may also be transiently introducedinto the recipient cell such that the introduced DNA molecule is notinherited by subsequent progeny and thus not considered “transgenic”.Accordingly, as used herein, a “non-transgenic” plant or plant cell is aplant which does not contain a foreign DNA stably integrated into itsgenome.

The term “plant promoter” as used herein is a promoter capable ofinitiating transcription in plant cells, whether or not its origin is aplant cell. Exemplary suitable plant promoters include, but are notlimited to, those that are obtained from plants, plant viruses, andbacteria such as Agrobacterium or Rhizobium which comprise genesexpressed in plant cells.

As used herein, a “fungal cell” refers to any type of eukaryotic cellwithin the kingdom of fungi. Phyla within the kingdom of fungi includeAscomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota,Glomeromycota, Microsporidia, and Neocallimastigomycota. Fungal cellsmay include yeasts, molds, and filamentous fungi. In some embodiments,the fungal cell is a yeast cell.

As used herein, the term “yeast cell” refers to any fungal cell withinthe phyla Ascomycota and Basidiomycota. Yeast cells may include buddingyeast cells, fission yeast cells, and mold cells. Without being limitedto these organisms, many types of yeast used in laboratory andindustrial settings are part of the phylum Ascomycota. In someembodiments, the yeast cell is an S. cerervisiae, Kluyveromycesmarxianus, or Issatchenkia orientalis cell. Other yeast cells mayinclude without limitation Candida spp. (e.g., Candida albicans),Yarrowia spp. (e.g., Yarrowia lipolytica), Pichia spp. (e.g., Pichiapastoris), Kluyveromyces spp. (e.g., Kluyveromyces lactis andKluyveromyces marxianus), Neurospora spp. (e.g., Neurospora crassa),Fusarium spp. (e.g., Fusarium oxysporum), and Issatchenkia spp. (e.g.,Issatchenkia orientalis, a.k.a. Pichia kudriavzevii and Candidaacidothermophilum). In some embodiments, the fungal cell is afilamentous fungal cell. As used herein, the term “filamentous fungalcell” refers to any type of fungal cell that grows in filaments, i.e.,hyphae or mycelia. Examples of filamentous fungal cells may includewithout limitation Aspergillus spp. (e.g., Aspergillus niger),Trichoderma spp. (e.g., Trichoderma reesei), Rhizopus spp. (e.g.,Rhizopus oryzae), and Mortierella spp. (e.g., Mortierella isabellina).

In some embodiments, the fungal cell is an industrial strain. As usedherein, “industrial strain” refers to any strain of fungal cell used inor isolated from an industrial process, e.g., production of a product ona commercial or industrial scale. Industrial strain may refer to afungal species that is typically used in an industrial process, or itmay refer to an isolate of a fungal species that may be also used fornon-industrial purposes (e.g., laboratory research). Examples ofindustrial processes may include fermentation (e.g., in production offood or beverage products), distillation, biofuel production, productionof a compound, and production of a polypeptide. Examples of industrialstrains may include, without limitation, JAY270 and ATCC4124.

In some embodiments, the fungal cell is a polyploid cell. As usedherein, a “polyploid” cell may refer to any cell whose genome is presentin more than one copy. A polyploid cell may refer to a type of cell thatis naturally found in a polyploid state, or it may refer to a cell thathas been induced to exist in a polyploid state (e.g., through specificregulation, alteration, inactivation, activation, or modification ofmeiosis, cytokinesis, or DNA replication). A polyploid cell may refer toa cell whose entire genome is polyploid, or it may refer to a cell thatis polyploid in a particular genomic locus of interest. Without wishingto be bound to theory, it is thought that the abundance of guide RNA maymore often be a rate-limiting component in genome engineering ofpolyploid cells than in haploid cells, and thus the methods using theCas13b CRISPR system described herein may take advantage of using acertain fungal cell type.

In some embodiments, the fungal cell is a diploid cell. As used herein,a “diploid” cell may refer to any cell whose genome is present in twocopies. A diploid cell may refer to a type of cell that is naturallyfound in a diploid state, or it may refer to a cell that has beeninduced to exist in a diploid state (e.g., through specific regulation,alteration, inactivation, activation, or modification of meiosis,cytokinesis, or DNA replication). For example, the S. cerevisiae strainS228C may be maintained in a haploid or diploid state. A diploid cellmay refer to a cell whose entire genome is diploid, or it may refer to acell that is diploid in a particular genomic locus of interest. In someembodiments, the fungal cell is a haploid cell. As used herein, a“haploid” cell may refer to any cell whose genome is present in onecopy. A haploid cell may refer to a type of cell that is naturally foundin a haploid state, or it may refer to a cell that has been induced toexist in a haploid state (e.g., through specific regulation, alteration,inactivation, activation, or modification of meiosis, cytokinesis, orDNA replication). For example, the S. cerevisiae strain S228C may bemaintained in a haploid or diploid state. A haploid cell may refer to acell whose entire genome is haploid, or it may refer to a cell that ishaploid in a particular genomic locus of interest.

As used herein, a “yeast expression vector” refers to a nucleic acidthat contains one or more sequences encoding an RNA and/or polypeptideand may further contain any desired elements that control the expressionof the nucleic acid(s), as well as any elements that enable thereplication and maintenance of the expression vector inside the yeastcell. Many suitable yeast expression vectors and features thereof areknown in the art; for example, various vectors and techniques areillustrated in in Yeast Protocols, 2nd edition, Xiao, W., ed. (HumanaPress, New York, 2007) and Buckholz, R. G. and Gleeson, M. A. (1991)Biotechnology (NY) 9(11): 1067-72. Yeast vectors may contain, withoutlimitation, a centromeric (CEN) sequence, an autonomous replicationsequence (ARS), a promoter, such as an RNA Polymerase III promoter,operably linked to a sequence or gene of interest, a terminator such asan RNA polymerase III terminator, an origin of replication, and a markergene (e.g., auxotrophic, antibiotic, or other selectable markers).Examples of expression vectors for use in yeast may include plasmids,yeast artificial chromosomes, 2μ plasmids, yeast integrative plasmids,yeast replicative plasmids, shuttle vectors, and episomal plasmids.

Stable Integration of RNA Targeting CRISPR System Components in theGenome of Plants and Plant Cells

In particular embodiments, it is envisaged that the polynucleotidesencoding the components of the RNA targeting CRISPR system areintroduced for stable integration into the genome of a plant cell. Inthese embodiments, the design of the transformation vector or theexpression system can be adjusted depending on when, where and underwhat conditions the guide RNA and/or the RNA targeting gene(s) areexpressed.

In particular embodiments, it is envisaged to introduce the componentsof the RNA targeting CRISPR system stably into the genomic DNA of aplant cell. Additionally or alternatively, it is envisaged to introducethe components of the RNA targeting CRISPR system for stable integrationinto the DNA of a plant organelle such as, but not limited to a plastid,e mitochondrion or a chloroplast.

The expression system for stable integration into the genome of a plantcell may contain one or more of the following elements: a promoterelement that can be used to express the guide RNA and/or RNA targetingenzyme in a plant cell; a 5′ untranslated region to enhance expression;an intron element to further enhance expression in certain cells, suchas monocot cells; a multiple-cloning site to provide convenientrestriction sites for inserting the one or more guide RNAs and/or theRNA targeting gene sequences and other desired elements; and a 3′untranslated region to provide for efficient termination of theexpressed transcript.

The elements of the expression system may be on one or more expressionconstructs which are either circular such as a plasmid or transformationvector, or non-circular such as linear double stranded DNA.

In a particular embodiment, a RNA targeting CRISPR expression systemcomprises at least:

-   (a) a nucleotide sequence encoding a guide RNA (gRNA) that    hybridizes with a target sequence in a plant, and wherein the guide    RNA comprises a guide sequence and a direct repeat sequence, and-   (b) a nucleotide sequence encoding a RNA targeting protein,    wherein components (a) or (b) are located on the same or on    different constructs, and whereby the different nucleotide sequences    can be under control of the same or a different regulatory element    operable in a plant cell.

DNA construct(s) containing the components of the RNA targeting CRISPRsystem, and, where applicable, template sequence may be introduced intothe genome of a plant, plant part, or plant cell by a variety ofconventional techniques. The process generally comprises the steps ofselecting a suitable host cell or host tissue, introducing theconstruct(s) into the host cell or host tissue, and regenerating plantcells or plants therefrom. In particular embodiments, the DNA constructmay be introduced into the plant cell using techniques such as but notlimited to electroporation, microinjection, aerosol beam injection ofplant cell protoplasts, or the DNA constructs can be introduced directlyto plant tissue using biolistic methods, such as DNA particlebombardment (see also Fu et al., Transgenic Res. 2000 February;9(1):11-9). The basis of particle bombardment is the acceleration ofparticles coated with gene/s of interest toward cells, resulting in thepenetration of the protoplasm by the particles and typically stableintegration into the genome. (see e.g. Klein et al, Nature (1987). Kleinet al, Bio/Technology (1992), Casas et al, Proc. Natl. Acad. Sci. USA(1993).).

In particular embodiments, the DNA constructs containing components ofthe RNA targeting CRISPR system may be introduced into the plant byAgrobacterium-mediated transformation. The DNA constructs may becombined with suitable T-DNA flanking regions and introduced into aconventional Agrobacterium tumeaciens host vector. The foreign DNA canbe incorporated into the genome of plants by infecting the plants or byincubating plant protoplasts with Agrobacterium bacteria, containing oneor more Ti (tumor-inducing) plasmids. (see e.g. Fraley et al., (1985),Rogers et al., (1987) and U.S. Pat. No. 5,563,055).

Plant Promoters

In order to ensure appropriate expression in a plant cell, thecomponents of the Cas13b CRISPR system described herein are typicallyplaced under control of a plant promoter, i.e. a promoter operable inplant cells. The use of different types of promoters is envisaged.

A constitutive plant promoter is a promoter that is able to express theopen reading frame (ORF) that it controls in all or nearly all of theplant tissues during all or nearly all developmental stages of the plant(referred to as “constitutive expression”). One non-limiting example ofa constitutive promoter is the cauliflower mosaic virus 35S promoter.The present invention envisages methods for modifying RNA sequences andas such also envisages regulating expression of plant biomolecules. Inparticular embodiments of the present invention it is thus advantageousto place one or more elements of the RNA targeting CRISPR system underthe control of a promoter that can be regulated. “Regulated promoter”refers to promoters that direct gene expression not constitutively, butin a temporally- and/or spatially-regulated manner, and includestissue-specific, tissue-preferred and inducible promoters. Differentpromoters may direct the expression of a gene in different tissues orcell types, or at different stages of development, or in response todifferent environmental conditions. In particular embodiments, one ormore of the RNA targeting CRISPR components are expressed under thecontrol of a constitutive promoter, such as the cauliflower mosaic virus35S promoter issue-preferred promoters can be utilized to targetenhanced expression in certain cell types within a particular planttissue, for instance vascular cells in leaves or roots or in specificcells of the seed. Examples of particular promoters for use in the RNAtargeting CRISPR system are found in Kawamata et al., (1997) Plant CellPhysiol 38:792-803; Yamamoto et al., (1997) Plant J 12:255-65; Hire etal, (1992) Plant Mol Biol 20:207-18, Kuster et al, (1995) Plant Mol Biol29:759-72, and Capana et al., (1994) Plant Mol Biol 25:681-91.

Examples of promoters that are inducible and that allow forspatiotemporal control of gene editing or gene expression may use a formof energy. The form of energy may include but is not limited to soundenergy, electromagnetic radiation, chemical energy and/or thermalenergy. Examples of inducible systems include tetracycline induciblepromoters (Tet-On or Tet-Off), small molecule two-hybrid transcriptionactivations systems (FKBP, ABA, etc), or light inducible systems(Phytochrome, LOV domains, or cryptochrome), such as a Light InducibleTranscriptional Effector (LITE) that direct changes in transcriptionalactivity in a sequence-specific manner. The components of a lightinducible system may include a RNA targeting CRISPR enzyme, alight-responsive cytochrome heterodimer (e.g. from Arabidopsisthaliana), and a transcriptional activation/repression domain. Furtherexamples of inducible DNA binding proteins and methods for their use areprovided in U.S. 61/736,465 and U.S. 61/721,283, which is herebyincorporated by reference in its entirety.

In particular embodiments, transient or inducible expression can beachieved by using, for example, chemical-regulated promotors, i.e.whereby the application of an exogenous chemical induces geneexpression. Modulating of gene expression can also be obtained by achemical-repressible promoter, where application of the chemicalrepresses gene expression. Chemical-inducible promoters include, but arenot limited to, the maize 1n2-2 promoter, activated by benzenesulfonamide herbicide safeners (De VeyIcier et al., (1997) Plant CellPhysiol 38:568-77), the maize GST promoter (GST-11-27, WO93/01294),activated by hydrophobic electrophilic compounds used as pre-emergentherbicides, and the tobacco PR-1 a promoter (Ono et al., (2004) BiosciBiotechnol Biochem 68:803-7) activated by salicylic acid. Promoterswhich are regulated by antibiotics, such as tetracycline-inducible andtetracycline-repressible promoters (Gatz et al., (1991) Mol Gen Genet227:229-37; U.S. Pat. Nos. 5,814,618 and 5,789,156) can also be usedherein.

Translocation to and/or Expression in Specific Plant Organelles

The expression system may comprise elements for translocation to and/orexpression in a specific plant organelle.

Chloroplast Targeting

In particular embodiments, it is envisaged that the RNA targeting CRISPRsystem is used to specifically modify expression and/or translation ofchloroplast genes or to ensure expression in the chloroplast. For thispurpose use is made of chloroplast transformation methods orcompartmentalization of the RNA targeting CRISPR components to thechloroplast. For instance, the introduction of genetic modifications inthe plastid genome can reduce biosafety issues such as gene flow throughpollen.

Methods of chloroplast transformation are known in the art and includeParticle bombardment, PEG treatment, and microinjection. Additionally,methods involving the translocation of transformation cassettes from thenuclear genome to the plastid can be used as described in WO2010061186.

Alternatively, it is envisaged to target one or more of the RNAtargeting CRISPR components to the plant chloroplast. This is achievedby incorporating in the expression construct a sequence encoding achloroplast transit peptide (CTP) or plastid transit peptide, operablylinked to the 5′ region of the sequence encoding the RNA targetingprotein. The CTP is removed in a processing step during translocationinto the chloroplast. Chloroplast targeting of expressed proteins iswell known to the skilled artisan (see for instance Protein Transportinto Chloroplasts, 2010, Annual Review of Plant Biology, Vol. 61:157-180). In such embodiments it is also desired to target the one ormore guide RNAs to the plant chloroplast. Methods and constructs whichcan be used for translocating guide RNA into the chloroplast by means ofa chloroplast localization sequence are described, for instance, in US20040142476, incorporated herein by reference. Such variations ofconstructs can be incorporated into the expression systems of theinvention to efficiently translocate the RNA targeting-guide RNA(s).

Introduction of Polynucleotides Encoding the CRISPR-RNA Targeting Systemin Algal Cells.

Transgenic algae (or other plants such as rape) may be particularlyuseful in the production of vegetable oils or biofuels such as alcohols(especially methanol and ethanol) or other products. These may beengineered to express or overexpress high levels of oil or alcohols foruse in the oil or biofuel industries.

U.S. Pat. No. 8,945,839 describes a method for engineering Micro-Algae(Chlamydomonas reinhardtii cells) species) using Cas9. Using similartools, the methods of the RNA targeting CRISPR system described hereincan be applied on Chlamydomonas species and other algae. In particularembodiments, RNA targeting protein and guide RNA(s) are introduced inalgae expressed using a vector that expresses RNA targeting proteinunder the control of a constitutive promoter such as Hsp70A-Rbc S2 orBeta2-tubulin. Guide RNA is optionally delivered using a vectorcontaining T7 promoter. Alternatively, RNA targeting mRNA and in vitrotranscribed guide RNA can be delivered to algal cells. Electroporationprotocols are available to the skilled person such as the standardrecommended protocol from the GeneArt Chlamydomonas Engineering kit.

Introduction of Polynucleotides Encoding RNA Targeting Components inYeast Cells

In particular embodiments, the invention relates to the use of the RNAtargeting CRISPR system for RNA editing in yeast cells. Methods fortransforming yeast cells which can be used to introduce polynucleotidesencoding the RNA targeting CRISPR system components are well known tothe artisan and are reviewed by Kawai et al., 2010, Bioeng Bugs. 2010November-December; 1(6): 395-403). Non-limiting examples includetransformation of yeast cells by lithium acetate treatment (which mayfurther include carrier DNA and PEG treatment), bombardment or byelectroporation.

Transient expression of RNA targeting CRISPR system components in plantsand plant cell

In particular embodiments, it is envisaged that the guide RNA and/or RNAtargeting gene are transiently expressed in the plant cell. In theseembodiments, the RNA targeting CRISPR system can ensure modification ofRNA target molecules only when both the guide RNA and the RNA targetingprotein is present in a cell, such that gene expression can further becontrolled. As the expression of the RNA targeting enzyme is transient,plants regenerated from such plant cells typically contain no foreignDNA. In particular embodiments the RNA targeting enzyme is stablyexpressed by the plant cell and the guide sequence is transientlyexpressed.

In particularly preferred embodiments, the RNA targeting CRISPR systemcomponents can be introduced in the plant cells using a plant viralvector (Scholthof et at 1996, Annu Rev Phytopathol. 1996; 34:299-323).In further particular embodiments, said viral vector is a vector from aDNA virus. For example, geminivirus (e.g., cabbage leaf curl virus, beanyellow dwarf virus, wheat dwarf virus, tomato leaf curl virus, maizestreak virus, tobacco leaf curl virus, or tomato golden mosaic virus) ornanovirus (e.g., Faba bean necrotic yellow virus). In other particularembodiments, said viral vector is a vector from an RNA virus. Forexample, tobravirus (e.g., tobacco rattle virus, tobacco mosaic virus),potexvirus (e.g., potato virus X), or hordeivirus (e.g., barley stripemosaic virus). The replicating genomes of plant viruses arenon-integrative vectors, which is of interest in the context of avoidingthe production of GMO plants.

In particular embodiments, the vector used for transient expression ofRNA targeting CRISPR constructs is for instance a pEAQ vector, which istailored for Agrobacterium-mediated transient expression (Sainsbury F.et al., Plant Biotechnol J. 2009 Sep., 7(7):682-93) in the protoplast.Precise targeting of genomic locations was demonstrated using a modifiedCabbage Leaf Curl virus (CaLCuV) vector to express gRNAs in stabletransgenic plants expressing a CRISPR enzyme (Scientific Reports 5,Article number: 14926 (2015), doi:10, 1038/srep14926).

In particular embodiments, double-stranded DNA fragments encoding theguide RNA and/or the RNA targeting gene can be transiently introducedinto the plant cell. In such embodiments, the introduced double-strandedDNA fragments are provided in sufficient quantity to modify RNAmolecule(s) in the cell but do not persist after a contemplated periodof time has passed or after one or more cell divisions. Methods fordirect DNA transfer in plants are known by the skilled artisan (see forinstance Davey et al. Plant Mol Biol. 1989 September; 13(3):273-85.)

In other embodiments, an RNA polynucleotide encoding the RNA targetingprotein is introduced into the plant cell, which is then translated andprocessed by the host cell generating the protein in sufficient quantityto modify the RNA molecule(s) cell (in the presence of at least oneguide RNA) but which does not persist after a contemplated period oftime has passed or after one or more cell divisions. Methods forintroducing mRNA to plant protoplasts for transient expression are knownby the skilled artisan (see for instance in Gallie, Plant Cell Reports(1993), 13; 119-122). Combinations of the different methods describedabove are also envisaged.

Delivery of RNA Targeting CRISPR Components to the Plant Cell

In particular embodiments, it is of interest to deliver one or morecomponents of the RNA targeting CRISPR system directly to the plantcell. This is of interest, inter alia, for the generation ofnon-transgenic plants (see below). In particular embodiments, one ormore of the RNA targeting components is prepared outside the plant orplant cell and delivered to the cell. For instance in particularembodiments, the RNA targeting protein is prepared in vitro prior tointroduction to the plant cell. RNA targeting protein can be prepared byvarious methods known by one of skill in the art and include recombinantproduction. After expression, the RNA targeting protein is isolated,refolded if needed, purified and optionally treated to remove anypurification tags, such as a His-tag. Once crude, partially purified, ormore completely purified RNA targeting protein is obtained, the proteinmay be introduced to the plant cell.

In particular embodiments, the RNA targeting protein is mixed with guideRNA targeting the RNA of interest to form a pre-assembledribonucleoprotein.

The individual components or pre-assembled ribonucleoprotein can beintroduced into the plant cell via electroporation, by bombardment withRNA targeting-associated gene product coated particles, by chemicaltransfection or by some other means of transport across a cell membrane.For instance, transfection of a plant protoplast with a pre-assembledCRISPR ribonucleoprotein has been demonstrated to ensure targetedmodification of the plant genome (as described by Woo et al. NatureBiotechnology, 2015; DOI: 10.1038/nbt.3389). These methods can bemodified to achieve targeted modification of RNA molecules in theplants.

In particular embodiments, the RNA targeting CRISPR system componentsare introduced into the plant cells using nanoparticles. The components,either as protein or nucleic acid or in a combination thereof, can beuploaded onto or packaged in nanoparticles and applied to the plants(such as for instance described in WO 2008042156 and US 20130185823). Inparticular, embodiments of the invention comprise nanoparticles uploadedwith or packed with DNA molecule(s) encoding the RNA targeting protein,DNA molecules encoding the guide RNA and/or isolated guide RNA asdescribed in WO2015089419.

Further means of introducing one or more components of the RNA targetingCRISPR system to the plant cell is by using cell penetrating peptides(CPP). Accordingly, in particular, embodiments the invention comprisescompositions comprising a cell penetrating peptide linked to an RNAtargeting protein. In particular embodiments of the present invention,an RNA targeting protein and/or guide RNA(s) is coupled to one or moreCPPs to effectively transport them inside plant protoplasts (Ramakrishna(2014, Genome Res. 2014 June; 24(6):1020-7 for Cas9 in human cells). Inother embodiments, the RNA targeting gene and/or guide RNA(s) areencoded by one or more circular or non-circular DNA molecule(s) whichare coupled to one or more CPPs for plant protoplast delivery. The plantprotoplasts are then regenerated to plant cells and further to plants.CPPs are generally described as short peptides of fewer than 35 aminoacids either derived from proteins or from chimeric sequences which arecapable of transporting biomolecules across cell membrane in a receptorindependent manner. CPP can be cationic peptides, peptides havinghydrophobic sequences, amphipatic peptides, peptides having proline-richand anti-microbial sequence, and chimeric or bipartite peptides (Poogaand Langel 2005). CPPs are able to penetrate biological membranes and assuch trigger the movement of various biomolecules across cell membranesinto the cytoplasm and to improve their intracellular routing, and hencefacilitate interaction of the biomolecule with the target. Examples ofCPP include amongst others: Tat, a nuclear transcriptional activatorprotein required for viral replication by HIV type1, penetratin, Kaposifibroblast growth factor (FGF) signal peptide sequence, integrin (33signal peptide sequence; polyarginine peptide Args sequence, Guaninerich-molecular transporters, sweet arrow peptide, etc. . . . .

Target RNA Envisaged for Plant, Algae or Fungal Applications

The target RNA, i.e. the RNA of interest, is the RNA to be targeted bythe present invention leading to the recruitment to, and the binding ofthe RNA targeting protein at, the target site of interest on the targetRNA. The target RNA may be any suitable form of RNA. This may include,in some embodiments, mRNA. In other embodiments, the target RNA mayinclude transfer RNA (tRNA) or ribosomal RNA (rRNA), In otherembodiments the target RNA may include interfering RNA (RNAi), microRNA(miRNA), microswitches, microzymes, satellite RNAs and RNA viruses. Thetarget RNA may be located in the cytoplasm of the plant cell, or in thecell nucleus or in a plant cell organelle such as a mitochondrion,chloroplast or plastid.

In particular embodiments, the RNA targeting CRISPR system is used tocleave RNA or otherwise inhibit RNA expression.

Use of RNA Targeting CRISPR System for Modulating Plant Gene ExpressionVia RNA Modulation

The RNA targeting protein may also be used, together with a suitableguide RNA, to target gene expression, via control of RNA processing. Thecontrol of RNA processing may include RNA processing reactions such asRNA splicing, including alternative splicing or specifically targetingcertain splice variants or isoforms; viral replication (in particular ofplant viruses, including virioids in plants and tRNA biosynthesis. TheRNA targeting protein in combination with a suitable guide RNA may alsobe used to control RNA activation (RNAa). RNAa leads to the promotion ofgene expression, so control of gene expression may be achieved that waythrough disruption or reduction of RNAa and thus less promotion of geneexpression.

The RNA targeting effector protein of the invention can further be usedfor antiviral activity in plants, in particular against RNA viruses. Theeffector protein can be targeted to the viral RNA using a suitable guideRNA selective for a selected viral RNA sequence. In particular, theeffector protein may be an active nuclease that cleaves RNA, such assingle stranded RNA. provided is therefore the use of an RNA targetingeffector protein of the invention as an antiviral agent. Examples ofviruses that can be counteracted in this way include, but are notlimited to, Tobacco mosaic virus (TMS′), Tomato spotted wilt virus(TSWV), Cucumber mosaic virus (CMV), Potato virus Y (PVY), Cauliflowermosaic virus (CaMV) (RT virus), Plum pox virus (PPV), Brome mosaic virus(BMV) and Potato virus X (PVX).

Examples of modulating RNA expression in plants, algae or fungi, as analternative of targeted gene modification are described herein further.

Of particular interest is the regulated control of gene expressionthrough regulated cleavage of mRNA. This can be achieved by placingelements of the RNA targeting under the control of regulated promotersas described herein.

Use of the RNA Targeting CRISPR System to Restore the Functionality oftRNA Molecules.

Pring et al describe RNA editing in plant mitochondria and chloroplaststhat alters mRNA sequences to code for different proteins than the DNA.(Plant Mol. Biol. (1993) 21 (6): 1163-1170. doi:10.1007/BF00023611). Inparticular embodiments of the invention, the elements of the RNAtargeting CRISPR system specifically targeting mitochondrial andchloroplast mRNA can be introduced in a plant or plant cell to expressdifferent proteins in such plant cell organelles mimicking the processesoccurring in vivo.

Use of the RNA Targeting CRISPR System as an Alternative to RNAInterference to Inhibit RNA Expression.

The RNA targeting CRISPR system has uses similar to RNA inhibition orRNA interference, thus can also be substituted for such methods. Inparticular embodiment, the methods of the present invention include theuse of the RNA targeting CRISPR as a substitute for e.g. an interferingribonucleic acid (such as an siRNA or shRNA or a dsRNA). Examples ofinhibition of RNA expression in plants, algae or fungi as an alternativeof targeted gene modification are described herein further.

Use of the RNA Targeting CRISPR System to Control RNA Interference.

Control over interfering RNA or miRNA may help reduce off-target effects(OTE) seen with those approaches by reducing the longevity of theinterfering RNA or miRNA in vivo or in vitro. In particular embodiments,the target RNA may include interfering RNA, i.e. RNA involved in an RNAinterference pathway, such as shRNA, siRNA and so forth. In otherembodiments, the target RNA may include microRNA (miRNA) or doublestranded RNA (dsRNA).

In other particular embodiments, if the RNA targeting protein andsuitable guide RNA(s) are selectively expressed (for example spatiallyor temporally under the control of a regulated promoter, for example atissue- or cell cycle-specific promoter and/or enhancer) this can beused to ‘protect’ the cells or systems (in vivo or in vitro) from RNAiin those cells. This may be useful in neighbouring tissues or cellswhere RNAi is not required or for the purposes of comparison of thecells or tissues where the effector protein and suitable guide are andare not expressed (i.e. where the RNAi is not controlled and where itis, respectively). The RNA targeting protein may be used to control orbind to molecules comprising or consisting of RNA, such as ribozymes,ribosomes or riboswitches. In embodiments of the invention, the guideRNA can recruit the RNA targeting protein to these molecules so that theRNA targeting protein is able to bind to them.

The RNA targeting CRISPR system of the invention can be applied in areasof in-planta RNAi technologies, without undue experimentation, from thisdisclosure, including insect pest management, plant disease managementand management of herbicide resistance, as well as in plant assay andfor other applications (see, for instance Kim et al., in PesticideBiochemistry and Physiology (Impact Factor: 2.01). January 2015; 120.DOI: 10.1016/j.pestbp.2015.01.002; Sharma et al. in Academic Journals(2015), Vol. 12(18) pp2303-2312); Green J. M, in Pest ManagementScience, Vol 70(9), pp 1351-1357), because the present applicationprovides the foundation for informed engineering of the system.

Use of RNA Targeting CRISPR System to Modify Riboswitches and ControlMetabolic Regulation in Plants, Algae and Fungi

Riboswitches (also known as aptozymes) are regulatory segments ofmessenger RNA that bind small molecules and in turn regulate geneexpression. This mechanism allows the cell to sense the intracellularconcentration of these small molecules. A particular riboswitchtypically regulates its adjacent gene by altering the transcription, thetranslation or the splicing of this gene. Thus, in particularembodiments of the present invention, control of riboswitch activity isenvisaged through the use of the RNA targeting protein in combinationwith a suitable guide RNA to target the riboswitch. This may be throughcleavage of, or binding to, the riboswitch. In particular embodiments,reduction of riboswitch activity is envisaged. Recently, a riboswitchthat binds thiamin pyrophosphate (TPP) was characterized and found toregulate thiamin biosynthesis in plants and algae. Furthermore itappears that this element is an essential regulator of primarymetabolism in plants (Bocobza and Aharoni, Plant J. 2014 August;79(4):693-703. doi: 10.1111/tpj.12540. Epub 2014 Jun. 17). TPPriboswitches are also found in certain fungi, such as in Neurosporacrassa, where it controls alternative splicing to conditionally producean Upstream Open Reading Frame (uORF), thereby affecting the expressionof downstream genes (Cheah M T et al., (2007) Nature 447 (7143):497-500. doi:10.1038/nature05769) The RNA targeting CRISPR systemdescribed herein may be used to manipulate the endogenous riboswitchactivity in plants, algae or fungi and as such alter the expression ofdownstream genes controlled by it. In particular embodiments, the RNAtargeting CRISPR system may be used in assaying riboswitch function invivo or in vitro and in studying its relevance for the metabolicnetwork. In particular embodiments the RNA targeting CRISPR system maypotentially be used for engineering of riboswitches as metabolitesensors in plants and platforms for gene control.

Use of RNA targeting CRISPR system in RNAi Screens for plants, algae orfungi

Identifying gene products whose knockdown is associated with phenotypicchanges, biological pathways can be interrogated and the constituentparts identified, via RNAi screens. In particular embodiments of theinvention, control may also be exerted over or during these screens byuse of the Cas13b protein and suitable guide RNA described herein toremove or reduce the activity of the RNAi in the screen and thusreinstate the activity of the (previously interfered with) gene product(by removing or reducing the interference/repression).

Use of RNA Targeting Proteins for Visualization of RNA Molecules In Vivoand In Vitro

In particular embodiments, the invention provides a nucleic acid bindingsystem. In situ hybridization of RNA with complementary probes is apowerful technique. Typically fluorescent DNA oligonucleotides are usedto detect nucleic acids by hybridization. Increased efficiency has beenattained by certain modifications, such as locked nucleic acids (LNAs),but there remains a need for efficient and versatile alternatives. Assuch, labelled elements of the RNA targeting system can be used as analternative for efficient and adaptable system for in situ hybridization

Further Applications of the RNA Targeting CRISPR System in Plants andYeasts Use of RNA Targeting CRISPR System in Biofuel Production

The term “biofuel” as used herein is an alternative fuel made from plantand plant-derived resources. Renewable biofuels can be extracted fromorganic matter whose energy has been obtained through a process ofcarbon fixation or are made through the use or conversion of biomass.This biomass can be used directly for biofuels or can be converted toconvenient energy containing substances by thermal conversion, chemicalconversion, and biochemical conversion. This biomass conversion canresult in fuel in solid, liquid, or gas form. There are two types ofbiofuels: bioethanol and biodiesel. Bioethanol is mainly produced by thesugar fermentation process of cellulose (starch), which is mostlyderived from maize and sugar cane. Biodiesel on the other hand is mainlyproduced from oil crops such as rapeseed, palm, and soybean. Biofuelsare used mainly for transportation.

Enhancing Plant Properties for Biofuel Production

In particular embodiments, the methods using the RNA targeting CRISPRsystem as described herein are used to alter the properties of the cellwall in order to facilitate access by key hydrolysing agents for a moreefficient release of sugars for fermentation. In particular embodiments,the biosynthesis of cellulose and/or lignin are modified. Cellulose isthe major component of the cell wall. The biosynthesis of cellulose andlignin are co-regulated. By reducing the proportion of lignin in a plantthe proportion of cellulose can be increased. In particular embodiments,the methods described herein are used to downregulate ligninbiosynthesis in the plant so as to increase fermentable carbohydrates.More particularly, the methods described herein are used to downregulateat least a first lignin biosynthesis gene selected from the groupconsisting of 4-coumarate 3-hydroxylase (C3H), phenylalanineammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), hydroxycinnamoyltransferase (HCT), caffeic acid O-methyltransferase (COMT), caffeoyl CoA3-O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H), cinnamylalcohol dehydrogenase (CAD), cinnamoyl CoA-reductase (CCR),4-coumarate-CoA ligase (4CL), monolignol-lignin-specificglycosyltransferase, and aldehyde dehydrogenase (ALDH) as disclosed inWO 2008064289 A2.

In particular embodiments, the methods described herein are used toproduce plant mass that produces lower levels of acetic acid duringfermentation (see also WO 2010096488).

Modifying Yeast for Biofuel Production

In particular embodiments, the RNA targeting enzyme provided herein isused for bioethanol production by recombinant micro-organisms. Forinstance, RNA targeting enzymes can be used to engineer micro-organisms,such as yeast, to generate biofuel or biopolymers from fermentablesugars and optionally to be able to degrade plant-derived lignocellulosederived from agricultural waste as a source of fermentable sugars. Moreparticularly, the invention provides methods whereby the RNA targetingCRISPR complex is used to modify the expression of endogenous genesrequired for biofuel production and/or to modify endogenous genes whymay interfere with the biofuel synthesis. More particularly the methodsinvolve stimulating the expression in a micro-organism such as a yeastof one or more nucleotide sequence encoding enzymes involved in theconversion of pyruvate to ethanol or another product of interest. Inparticular embodiments the methods ensure the stimulation of expressionof one or more enzymes which allows the micro-organism to degradecellulose, such as a cellulase. In yet further embodiments, the RNAtargeting CRISPR complex is used to suppress endogenous metabolicpathways which compete with the biofuel production pathway.

Modifying Algae and Plants for Production of Vegetable Oils or Biofuels

Transgenic algae or other plants such as rape may be particularly usefulin the production of vegetable oils or biofuels such as alcohols(especially methanol and ethanol), for instance. These may be engineeredto express or overexpress high levels of oil or alcohols for use in theoil or biofuel industries.

U.S. Pat. No. 8,945,839 describes a method for engineering Micro-Algae(Chlamydomonas reinhardtii cells) using Cas9. Using similar tools, themethods of the RNA targeting CRISPR system described herein can beapplied on Chlamydomonas species and other algae. In particularembodiments, the RNA targeting effector protein and guide RNA areintroduced in algae expressed using a vector that expresses the RNAtargeting effector protein under the control of a constitutive promotersuch as Hsp70A-Rbc S2 or Beta2-tubulin. Guide RNA will be deliveredusing a vector containing T7 promoter. Alternatively, in vitrotranscribed guide RNA can be delivered to algae cells. Electroporationprotocol follows standard recommended protocol from the GeneArtChlamydomonas Engineering kit.

Particular Applications of the RNA Targeting Enzymes in Plants

In particular embodiments, present invention can be used as a therapyfor virus removal in plant systems as it is able to cleave viral RNA.Previous studies in human systems have demonstrated the success ofutilizing CRISPR in targeting the single strand RNA virus, hepatitis C(A. Price, et al., Proc. Natl. Acad. Sci, 2015). These methods may alsobe adapted for using the RNA targeting CRISPR system in plants.

Improved Plants

The present invention also provides plants and yeast cells obtainableand obtained by the methods provided herein. The improved plantsobtained by the methods described herein may be useful in food or feedproduction through the modified expression of genes which, for instanceensure tolerance to plant pests, herbicides, drought, low or hightemperatures, excessive water, etc.

The improved plants obtained by the methods described herein, especiallycrops and algae may be useful in food or feed production throughexpression of, for instance, higher protein, carbohydrate, nutrient orvitamin levels than would normally be seen in the wildtype. In thisregard, improved plants, especially pulses and tubers are preferred.

Improved algae or other plants such as rape may be particularly usefulin the production of vegetable oils or biofuels such as alcohols(especially methanol and ethanol), for instance. These may be engineeredto express or overexpress high levels of oil or alcohols for use in theoil or biofuel industries.

The invention also provides for improved parts of a plant. Plant partsinclude, but are not limited to, leaves, stems, roots, tubers, seeds,endosperm, ovule, and pollen. Plant parts as envisaged herein may beviable, nonviable, regeneratable, and/or non-regeneratable.

It is also encompassed herein to provide plant cells and plantsgenerated according to the methods of the invention. Gametes, seeds,embryos, either zygotic or somatic, progeny or hybrids of plantscomprising the genetic modification, which are produced by traditionalbreeding methods, are also included within the scope of the presentinvention. Such plants may contain a heterologous or foreign DNAsequence inserted at or instead of a target sequence. Alternatively,such plants may contain only an alteration (mutation, deletion,insertion, substitution) in one or more nucleotides. As such, suchplants will only be different from their progenitor plants by thepresence of the particular modification.

In an embodiment of the invention, a Cas13b system is used to engineerpathogen resistant plants, for example by creating resistance againstdiseases caused by bacteria, fungi or viruses. In certain embodiments,pathogen resistance can be accomplished by engineering crops to producea Cas13b system that wil be ingested by an insect pest, leading tomortality. In an embodiment of the invention, a Cas13b system is used toengineer abiotic stress tolerance. In another embodiment, a Cas13bsystem is used to engineer drought stress tolerance or salt stresstolerance, or cold or heat stress tolerance. Younis et al. 2014, Int. J.Biol. Sci. 10; 1150 reviewed potential targets of plant breedingmethods, all of which are amenable to correction or improvement throughuse of a Cas13b system described herein. Some non-limiting target cropsinclude Oryza sativa L, Prunus domestica L., Gossypium hirsutum,Nicotiana rustica, Zea mays, Medicago sativa, Nicotiana benthamiana andArabidopsis thaliana

In an embodiment of the invention, a Cas13b system is used formanagement of crop pests. For example, a Cas13b system operable in acrop pest can be expressed from a plant host or transferred directly tothe target, for example using a viral vector.

In an embodiment, the invention provides a method of efficientlyproducing homozygous organisms from a heterozygous non-human startingorganism. In an embodiment, the invention is used in plant breeding. Inanother embodiment, the invention is used in animal breeding. In suchembodiments, a homozygous organism such as a plant or animal is made bypreventing or suppressing recombination by interfering with at least onetarget gene involved in double strand breaks, chromosome pairing and/orstrand exchange.

Application of the Cas13b Proteins in Optimized Functional RNA TargetingSystems

In an aspect the invention provides a system for specific delivery offunctional components to the RNA environment. This can be ensured usingthe CRISPR systems comprising the RNA targeting effector proteins of thepresent invention which allow specific targeting of different componentsto RNA. More particularly such components include activators orrepressors, such as activators or repressors of RNA translation,degradation, etc. Applications of this system are described elsewhereherein.

According to one aspect the invention provides non-naturally occurringor engineered composition comprising a guide RNA comprising a guidesequence capable of hybridizing to a target sequence in a genomic locusof interest in a cell, wherein the guide RNA is modified by theinsertion of one or more distinct RNA sequence(s) that bind an adaptorprotein. In particular embodiments, the RNA sequences may bind to two ormore adaptor proteins (e.g. aptamers), and wherein each adaptor proteinis associated with one or more functional domains. The guide RNAs of theCas13b enzymes described herein are shown to be amenable to modificationof the guide sequence. In particular embodiments, the guide RNA ismodified by the insertion of distinct RNA sequence(s) 5′ of the directrepeat, within the direct repeat, or 3′ of the guide sequence. Whenthere is more than one functional domain, the functional domains can besame or different, e.g., two of the same or two different activators orrepressors. In an aspect the invention provides a herein-discussedcomposition, wherein the one or more functional domains are attached tothe RNA targeting enzyme so that upon binding to the target RNA thefunctional domain is in a spatial orientation allowing for thefunctional domain to function in its attributed function; In an aspectthe invention provides a herein-discussed composition, wherein thecomposition comprises a CRISPR-Cas complex having at least threefunctional domains, at least one of which is associated with the RNAtargeting enzyme and at least two of which are associated with the gRNA.

Accordingly, in an aspect the invention provides non-naturally occurringor engineered CRISPR-Cas13b complex composition comprising the guide RNAas herein-discussed and a Cas13b which is an RNA targeting enzyme,wherein optionally the RNA targeting enzyme comprises at least onemutation, such that the RNA targeting enzyme has no more than 5% of thenuclease activity of the enzyme not having the at least one mutation,and optionally one or more comprising at least one or more nuclearlocalization sequences. In particular embodiments, the guide RNA isadditionally or alternatively modified so as to still ensure binding ofthe RNA targeting enzyme but to prevent cleavage by the RNA targetingenzyme (as detailed elsewhere herein).

In particular embodiments, the RNA targeting enzyme is a Cas13b enzymewhich has a diminished nuclease activity of at least 97%, or 100% ascompared with the Cas13b enzyme not having the at least one mutation. Inan aspect the invention provides a herein-discussed composition, whereinthe Cas13b enzyme comprises two or more mutations as otherwiseherein-discussed.

In particular embodiments, an RNA targeting system is provided asdescribed herein above comprising two or more functional domains. Inparticular embodiments, the two or more functional domains areheterologous functional domain. In particular embodiments, the systemcomprises an adaptor protein which is a fusion protein comprising afunctional domain, the fusion protein optionally comprising a linkerbetween the adaptor protein and the functional domain. In particularembodiments, the linker includes a GlySer linker. Additionally oralternatively, one or more functional domains are attached to the RNAeffector protein by way of a linker, optionally a GlySer linker. Inparticular embodiments, the one or more functional domains are attachedto the RNA targeting enzyme through one or both of the HEPN domains.

In an aspect the invention provides a herein-discussed composition,wherein the one or more functional domains associated with the adaptorprotein or the RNA targeting enzume is a domain capable of activating orrepressing RNA translation. In an aspect the invention provides aherein-discussed composition, wherein at least one of the one or morefunctional domains associated with the adaptor protein have one or moreactivities comprising methylase activity, demethylase activity,transcription activation activity, transcription repression activity,transcription release factor activity, histone modification activity,DNA integration activity RNA cleavage activity, DNA cleavage activity ornucleic acid binding activity, or molecular switch activity or chemicalinducibility or light inducibility.

In an aspect the invention provides a herein-discussed compositioncomprising an aptamer sequence. In particular embodiments, the aptamersequence is two or more aptamer sequences specific to the same adaptorprotein. In an aspect the invention provides a herein-discussedcomposition, wherein the aptamer sequence is two or more aptamersequences specific to different adaptor protein. In an aspect theinvention provides a herein-discussed composition, wherein the adaptorprotein comprises MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34,JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5,ϕCb8r, ϕCb 12r, ϕCb23r, 7s, PRR1. Accordingly, in particularembodiments, the aptamer is selected from a binding protein specificallybinding any one of the adaptor proteins listed above. In an aspect theinvention provides a herein-discussed composition, wherein the cell is aeukaryotic cell. In an aspect the invention provides a herein-discussedcomposition, wherein the eukaryotic cell is a mammalian cell, a plantcell or a yeast cell, whereby the mammalian cell is optionally a mousecell. In an aspect the invention provides a herein-discussedcomposition, wherein the mammalian cell is a human cell.

In an aspect the invention provides a herein above-discussed compositionwherein there is more than one gRNA, and the gRNAs target differentsequences whereby when the composition is employed, there ismultiplexing. In an aspect the invention provides a composition whereinthere is more than one gRNA modified by the insertion of distinct RNAsequence(s) that bind to one or more adaptor proteins.

In an aspect the invention provides a herein-discussed compositionwherein one or more adaptor proteins associated with one or morefunctional domains is present and bound to the distinct RNA sequence(s)inserted into the guide RNA(s).

In an aspect the invention provides a herein-discussed compositionwherein the guide RNA is modified to have at least one non-codingfunctional loop; e.g., wherein the at least one non-coding functionalloop is repressive; for instance, wherein at least one non-codingfunctional loop comprises Alu.

In an aspect the invention provides a method for modifying geneexpression comprising the administration to a host or expression in ahost in vivo of one or more of the compositions as herein-discussed.

In an aspect the invention provides a herein-discussed method comprisingthe delivery of the composition or nucleic acid molecule(s) codingtherefor, wherein said nucleic acid molecule(s) are operatively linkedto regulatory sequence(s) and expressed in vivo. In an aspect theinvention provides a herein-discussed method wherein the expression invivo is via a lentivirus, an adenovirus, or an AAV.

In an aspect the invention provides a mammalian cell line of cells asherein-discussed, wherein the cell line is, optionally, a human cellline or a mouse cell line. In an aspect the invention provides atransgenic mammalian model, optionally a mouse, wherein the model hasbeen transformed with a herein-discussed composition or is a progeny ofsaid transformant.

In an aspect the invention provides a nucleic acid molecule(s) encodingguide RNA or the RNA targeting CRISPR-Cas complex or the composition asherein-discussed. In an aspect the invention provides a vectorcomprising: a nucleic acid molecule encoding a guide RNA (gRNA)comprising a guide sequence capable of hybridizing to a target sequencein a genomic locus of interest in a cell, wherein the direct repeat ofthe gRNA is modified by the insertion of distinct RNA sequence(s) thatbind(s) to two or more adaptor proteins, and wherein each adaptorprotein is associated with one or more functional domains; or, whereinthe gRNA is modified to have at least one non-coding functional loop. Inan aspect the invention provides vector(s) comprising nucleic acidmolecule(s) encoding: non-naturally occurring or engineered CRISPR-Cascomplex composition comprising the gRNA herein-discussed, and an RNAtargeting enzyme, wherein optionally the RNA targeting enzyme comprisesat least one mutation, such that the RNA targeting enzyme has no morethan 5% of the nuclease activity of the RNA targeting enzyme not havingthe at least one mutation, and optionally one or more comprising atleast one or more nuclear localization sequences. In an aspect a vectorcan further comprise regulatory element(s) operable in a eukaryotic celloperably linked to the nucleic acid molecule encoding the guide RNA(gRNA) and/or the nucleic acid molecule encoding the RNA targetingenzyme and/or the optional nuclear localization sequence(s).

In one aspect, the invention provides a kit comprising one or more ofthe components described hereinabove. In some embodiments, the kitcomprises a vector system as described above and instructions for usingthe kit.

In an aspect the invention provides a method of screening for gain offunction (GOF) or loss of function (LOF) or for screening non-codingRNAs or potential regulatory regions (e.g. enhancers, repressors)comprising the cell line of as herein-discussed or cells of the modelherein-discussed containing or expressing the RNA targeting enzyme andintroducing a composition as herein-discussed into cells of the cellline or model, whereby the gRNA includes either an activator or arepressor, and monitoring for GOF or LOF respectively as to those cellsas to which the introduced gRNA includes an activator or as to thosecells as to which the introduced gRNA includes a repressor.

In an aspect the invention provides a library of non-naturally occurringor engineered compositions, each comprising a RNA targeting CRISPR guideRNA (gRNA) comprising a guide sequence capable of hybridizing to atarget RNA sequence of interest in a cell, an RNA targeting enzyme,wherein the RNA targeting enzyme comprises at least one mutation, suchthat the RNA targeting enzyme has no more than 5% of the nucleaseactivity of the RNA targeting enzyme not having the at least onemutation, wherein the gRNA is modified by the insertion of distinct RNAsequence(s) that bind to one or more adaptor proteins, and wherein theadaptor protein is associated with one or more functional domains,wherein the composition comprises one or more or two or more adaptorproteins, wherein the each protein is associated with one or morefunctional domains, and wherein the gRNAs comprise a genome wide librarycomprising a plurality of RNA targeting guide RNAs (gRNAs). In an aspectthe invention provides a library as herein-discussed, wherein the RNAtargeting RNA targeting enzyme has a diminished nuclease activity of atleast 97%, or 100% as compare with the RNA targeting enzyme not havingthe at least one mutation. In an aspect the invention provides a libraryas herein-discussed, wherein the adaptor protein is a fusion proteincomprising the functional domain. In an aspect the invention provides alibrary as herein discussed, wherein the gRNA is not modified by theinsertion of distinct RNA sequence(s) that bind to the one or two ormore adaptor proteins. In an aspect the invention provides a library asherein discussed, wherein the one or two or more functional domains areassociated with the RNA targeting enzyme. In an aspect the inventionprovides a library as herein discussed, wherein the cell population ofcells is a population of eukaryotic cells. In an aspect the inventionprovides a library as herein discussed, wherein the eukaryotic cell is amammalian cell, a plant cell or a yeast cell. In an aspect the inventionprovides a library as herein discussed, wherein the mammalian cell is ahuman cell. In an aspect the invention provides a library as hereindiscussed, wherein the population of cells is a population of embryonicstem (ES) cells.

In an aspect the invention provides a library as herein discussed,wherein the targeting is of about 100 or more RNA sequences. In anaspect the invention provides a library as herein discussed, wherein thetargeting is of about 1000 or more RNA sequences. In an aspect theinvention provides a library as herein discussed, wherein the targetingis of about 20,000 or more sequences. In an aspect the inventionprovides a library as herein discussed, wherein the targeting is of theentire transcriptome. In an aspect the invention provides a library asherein discussed, wherein the targeting is of a panel of targetsequences focused on a relevant or desirable pathway. In an aspect theinvention provides a library as herein discussed, wherein the pathway isan immune pathway. In an aspect the invention provides a library asherein discussed, wherein the pathway is a cell division pathway.

In one aspect, the invention provides a method of generating a modeleukaryotic cell comprising a gene with modified expression. In someembodiments, a disease gene is any gene associated an increase in therisk of having or developing a disease. In some embodiments, the methodcomprises (a) introducing one or more vectors encoding the components ofthe system described herein above into a eukaryotic cell, and (b)allowing a CRISPR complex to bind to a target polynucleotide so as tomodify expression of a gene, thereby generating a model eukaryotic cellcomprising modified gene expression.

The structural information provided herein allows for interrogation ofguide RNA interaction with the target RNA and the RNA targeting enzymepermitting engineering or alteration of guide RNA structure to optimizefunctionality of the entire RNA targeting CRISPR-Cas system. Forexample, the guide RNA may be extended, without colliding with the RNAtargeting protein by the insertion of adaptor proteins that can bind toRNA. These adaptor proteins can further recruit effector proteins orfusions which comprise one or more functional domains.

An aspect of the invention is that the above elements are comprised in asingle composition or comprised in individual compositions. Thesecompositions may advantageously be applied to a host to elicit afunctional effect on the genomic level.

The skilled person will understand that modifications to the guide RNAwhich allow for binding of the adapter+functional domain but not properpositioning of the adapter+functional domain (e.g. due to sterichindrance within the three dimensional structure of the CRISPR complex)are modifications which are not intended. The one or more modified guideRNA may be modified, by introduction of a distinct RNA sequence(s) 5′ ofthe direct repeat, within the direct repeat, or 3′ of the guidesequence.

The modified guide RNA, the inactivated RNA targeting enzyme (with orwithout functional domains), and the binding protein with one or morefunctional domains, may each individually be comprised in a compositionand administered to a host individually or collectively. Alternatively,these components may be provided in a single composition foradministration to a host. Administration to a host may be performed viaviral vectors known to the skilled person or described herein fordelivery to a host (e.g. lentiviral vector, adenoviral vector, AAVvector). As explained herein, use of different selection markers (e.g.for lentiviral gRNA selection) and concentration of gRNA (e.g. dependenton whether multiple gRNAs are used) may be advantageous for eliciting animproved effect.

Using the provided compositions, the person skilled in the art canadvantageously and specifically target single or multiple loci with thesame or different functional domains to elicit one or more genomicevents. The compositions may be applied in a wide variety of methods forscreening in libraries in cells and functional modeling in vivo (e.g.gene activation of lincRNA and indentification of function;gain-of-function modeling; loss-of-function modeling; the use thecompositions of the invention to establish cell lines and transgenicanimals for optimization and screening purposes).

The current invention comprehends the use of the compositions of thecurrent invention to establish and utilize conditional or inducibleCRISPR RNA targeting events. (See, e.g., Platt et al., Cell (2014),http://dx.doi.org/10.1016/j.cell.2014.09.014, or PCT patent publicationscited herein, such as WO 2014/093622 (PCT/US2013/074667), which are notbelieved prior to the present invention or application). For example,the target cell comprises RNA targeting CRISPR enzyme conditionally orinducibly (e.g. in the form of Cre dependent constructs) and/or theadapter protein conditionally or inducibly and, on expression of avector introduced into the target cell, the vector expresses that whichinduces or gives rise to the condition of s RNA targeting enzymeexpression and/or adaptor expression in the target cell. By applying theteaching and compositions of the current invention with the known methodof creating a CRISPR complex, inducible gene expression affected byfunctional domains are also an aspect of the current invention.Alternatively, the adaptor protein may be provided as a conditional orinducible element with a conditional or inducible s RNA targeting enzymeto provide an effective model for screening purposes, whichadvantageously only requires minimal design and administration ofspecific gRNAs for a broad number of applications.

Guide RNA According to the Invention Comprising a Dead Guide Sequence

In one aspect, the invention provides guide sequences which are modifiedin a manner which allows for formation of the CRISPR complex andsuccessful binding to the target, while at the same time, not allowingfor successful nuclease activity (i.e. without nuclease activity/withoutindel activity). For matters of explanation such modified guidesequences are referred to as “dead guides” or “dead guide sequences”.These dead guides or dead guide sequences can be thought of ascatalytically inactive or conformationally inactive with regard tonuclease activity. Indeed, dead guide sequences may not sufficientlyengage in productive base pairing with respect to the ability to promotecatalytic activity or to distinguish on-target and off-target bindingactivity. Briefly, the assay involves synthesizing a CRISPR target RNAand guide RNAs comprising mismatches with the target RNA, combiningthese with the RNA targeting enzyme and analyzing cleavage based on gelsbased on the presence of bands generated by cleavage products, andquantifying cleavage based upon relative band intensities.

Hence, in a related aspect, the invention provides a non-naturallyoccurring or engineered composition RNA targeting CRISPR-Cas systemcomprising a functional RNA targeting as described herein, and guide RNA(gRNA) wherein the gRNA comprises a dead guide sequence whereby the gRNAis capable of hybridizing to a target sequence such that the RNAtargeting CRISPR-Cas system is directed to a genomic locus of interestin a cell without detectable RNA cleavage activity of a non-mutant RNAtargeting enzyme of the system. It is to be understood that any of thegRNAs according to the invention as described herein elsewhere may beused as dead gRNAs/gRNAs comprising a dead guide sequence as describedherein below. Any of the methods, products, compositions and uses asdescribed herein elsewhere is equally applicable with the deadgRNAs/gRNAs comprising a dead guide sequence as further detailed below.By means of further guidance, the following particular aspects andembodiments are provided.

The ability of a dead guide sequence to direct sequence-specific bindingof a CRISPR complex to an RNA target sequence may be assessed by anysuitable assay. For example, the components of a CRISPR systemsufficient to form a CRISPR complex, including the dead guide sequenceto be tested, may be provided to a host cell having the correspondingtarget sequence, such as by transfection with vectors encoding thecomponents of the CRISPR sequence, followed by an assessment ofpreferential cleavage within the target sequence. For instance, cleavageof a target RNA polynucleotide sequence may be evaluated in a test tubeby providing the target sequence, components of a CRISPR complex,including the dead guide sequence to be tested and a control guidesequence different from the test dead guide sequence, and comparingbinding or rate of cleavage at the target sequence between the test andcontrol guide sequence reactions. Other assays are possible, and willoccur to those skilled in the art. A dead guide sequence may be selectedto target any target sequence. In some embodiments, the target sequenceis a sequence within a genome of a cell.

As explained further herein, several structural parameters allow for aproper framework to arrive at such dead guides. Dead guide sequences aretypically shorter than respective guide sequences which result in activeRNA cleavage. In particular embodiments, dead guides are 5%, 10%, 20%,30%, 40%, 50%, shorter than respective guides directed to the same.

As explained below and known in the art, one aspect of gRNA-RNAtargeting specificity is the direct repeat sequence, which is to beappropriately linked to such guides. In particular, this implies thatthe direct repeat sequences are designed dependent on the origin of theRNA targeting enzyme. Thus, structural data available for validated deadguide sequences may be used for designing Cas13b specific equivalents.Structural similarity between, e.g., the orthologous nuclease domainsHEPN of two or more Cas13b effector proteins may be used to transferdesign equivalent dead guides. Thus, the dead guide herein may beappropriately modified in length and sequence to reflect such Cas13bspecific equivalents, allowing for formation of the CRISPR complex andsuccessful binding to the target RNA, while at the same time, notallowing for successful nuclease activity.

The use of dead guides in the context herein as well as the state of theart provides a surprising and unexpected platform for network biologyand/or systems biology in both in vitro, ex vivo, and in vivoapplications, allowing for multiplex gene targeting, and in particularbidirectional multiplex gene targeting. Prior to the use of dead guides,addressing multiple targets has been challenging and in some cases notpossible. With the use of dead guides, multiple targets, and thusmultiple activities, may be addressed, for example, in the same cell, inthe same animal, or in the same patient. Such multiplexing may occur atthe same time or staggered for a desired timeframe.

For example, the dead guides allow to use gRNA as a means for genetargeting, without the consequence of nuclease activity, while at thesame time providing directed means for activation or repression. GuideRNA comprising a dead guide may be modified to further include elementsin a manner which allow for activation or repression of gene activity,in particular protein adaptors (e.g. aptamers) as described hereinelsewhere allowing for functional placement of gene effectors (e.g.activators or repressors of gene activity). One example is theincorporation of aptamers, as explained herein and in the state of theart. By engineering the gRNA comprising a dead guide to incorporateprotein-interacting aptamers (Konermann et al., “Genome-scaletranscription activation by an engineered CRISPR-Cas9 complex,”doi:10.1038/nature14136, incorporated herein by reference), one mayassemble multiple distinct effector domains. Such may be modeled afternatural processes.

General Provisions

In an aspect, the invention provides a nucleic acid binding system. Insitu hybridization of RNA with complementary probes is a powerfultechnique. Typically fluorescent DNA oligonucleotides are used to detectnucleic acids by hybridization. Increased efficiency has been attainedby certain modifications, such as locked nucleic acids (LNAs), but thereremains a need for efficient and versatile alternatives. The inventionprovides an efficient and adaptable system for in situ hybridization.

In embodiments of the invention the terms guide sequence and guide RNAare used interchangeably as in foregoing cited documents such as WO2014/093622 (PCT/US2013/074667). In general, a guide sequence is anypolynucleotide sequence having sufficient complementarity with a targetpolynucleotide sequence to hybridize with the target sequence and directsequence-specific binding of a CRISPR complex to the target sequence. Insome embodiments, the degree of complementarity between a guide sequenceand its corresponding target sequence, when optimally aligned using asuitable alignment algorithm, is about or more than about 50%, 60%, 75%,80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may bedetermined with the use of any suitable algorithm for aligningsequences, non-limiting example of which include the Smith-Watermanalgorithm, the Needleman-Wunsch algorithm, algorithms based on theBurrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW,Clustal X, BLAT, Novoalign (Novocraft Technologies; available atwww.novocraft.com), ELAND (Illumina, San Diego, Calif.), SOAP (availableat soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). Insome embodiments, a guide sequence is about or more than about 5, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In someembodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30,25, 20, 15, 12, or fewer nucleotides in length. Preferably the guidesequence is 10-30 nucleotides long. The ability of a guide sequence todirect sequence-specific binding of a CRISPR complex to a targetsequence may be assessed by any suitable assay. For example, thecomponents of a CRISPR system sufficient to form a CRISPR complex,including the guide sequence to be tested, may be provided to a hostcell having the corresponding target sequence, such as by transfectionwith vectors encoding the components of the CRISPR sequence, followed byan assessment of preferential cleavage within the target sequence, suchas by Surveyor assay as described herein. Similarly, cleavage of atarget polynucleotide sequence may be evaluated in a test tube byproviding the target sequence, components of a CRISPR complex, includingthe guide sequence to be tested and a control guide sequence differentfrom the test guide sequence, and comparing binding or rate of cleavageat the target sequence between the test and control guide sequencereactions. Other assays are possible, and will occur to those skilled inthe art. A guide sequence may be selected to target any target sequence.In some embodiments, the target sequence is a sequence within a genomeof a cell. Exemplary target sequences include those that are unique inthe target genome.

In general, and throughout this specification, the term “vector” refersto a nucleic acid molecule capable of transporting another nucleic acidto which it has been linked. Vectors include, but are not limited to,nucleic acid molecules that are single-stranded, double-stranded, orpartially double-stranded; nucleic acid molecules that comprise one ormore free ends, no free ends (e.g., circular); nucleic acid moleculesthat comprise DNA, RNA, or both; and other varieties of polynucleotidesknown in the art. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments canbe inserted, such as by standard molecular cloning techniques. Anothertype of vector is a viral vector, wherein virally-derived DNA or RNAsequences are present in the vector for packaging into a virus (e.g.,retroviruses, replication defective retroviruses, adenoviruses,replication defective adenoviruses, and adeno-associated viruses). Viralvectors also include polynucleotides carried by a virus for transfectioninto a host cell. Certain vectors are capable of autonomous replicationin a host cell into which they are introduced (e.g., bacterial vectorshaving a bacterial origin of replication and episomal mammalianvectors). Other vectors (e.g., non-episomal mammalian vectors) areintegrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively-linked. Such vectors are referred toherein as “expression vectors.” Vectors for and that result inexpression in a eukaryotic cell can be referred to herein as “eukaryoticexpression vectors.” Common expression vectors of utility in recombinantDNA techniques are often in the form of plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.,in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell).

The term “regulatory element” is intended to include promoters,enhancers, internal ribosomal entry sites (IRES), and other expressioncontrol elements (e.g., transcription termination signals, such aspolyadenylation signals and poly-U sequences). Such regulatory elementsare described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).Regulatory elements include those that direct constitutive expression ofa nucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). A tissue-specific promoter maydirect expression primarily in a desired tissue of interest, such asmuscle, neuron, bone, skin, blood, specific organs (e.g., liver,pancreas), or particular cell types (e.g., lymphocytes). Regulatoryelements may also direct expression in a temporal-dependent manner, suchas in a cell-cycle dependent or developmental stage-dependent manner,which may or may not also be tissue or cell-type specific. In someembodiments, a vector comprises one or more pol III promoter (e.g., 1,2, 3, 4, 5, or more pol III promoters), one or more pol II promoters(e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol Ipromoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), orcombinations thereof. Examples of pol III promoters include, but are notlimited to, U6 and H1 promoters. Examples of pol II promoters include,but are not limited to, the retroviral Rous sarcoma virus (RSV) LTRpromoter (optionally with the RSV enhancer), the cytomegalovirus (CMV)promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al,Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductasepromoter, the β-actin promoter, the phosphoglycerol kinase (PGK)promoter, and the EF1α promoter. Also encompassed by the term“regulatory element” are enhancer elements, such as WPRE; CMV enhancers;the R-U5′ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p.466-472, 1988); SV40 enhancer; and the intron sequence between exons 2and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p.1527-31, 1981). It will be appreciated by those skilled in the art thatthe design of the expression vector can depend on such factors as thechoice of the host cell to be transformed, the level of expressiondesired, etc. A vector can be introduced into host cells to therebyproduce transcripts, proteins, or peptides, including fusion proteins orpeptides, encoded by nucleic acids as described herein (e.g., clusteredregularly interspersed short palindromic repeats (CRISPR) transcripts,proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).

Advantageous vectors include lentiviruses and adeno-associated viruses,and types of such vectors can also be selected for targeting particulartypes of cells.

As used herein, the term “crRNA” or “guide RNA” or “single guide RNA” or“sgRNA” or “one or more nucleic acid components” of a Type V or Type VICRISPR-Cas locus effector protein comprises any polynucleotide sequencehaving sufficient complementarity with a target nucleic acid sequence tohybridize with the target nucleic acid sequence and directsequence-specific binding of a nucleic acid-targeting complex to thetarget nucleic acid sequence.

In certain embodiments, the CRISPR system as provided herein can makeuse of a crRNA or analogous polynucleotide comprising a guide sequence,wherein the polynucleotide is an RNA, a DNA or a mixture of RNA and DNA,and/or wherein the polynucleotide comprises one or more nucleotideanalogs. The sequence can comprise any structure, including but notlimited to a structure of a native crRNA, such as a bulge, a hairpin ora stem loop structure. In certain embodiments, the polynucleotidecomprising the guide sequence forms a duplex with a secondpolynucleotide sequence which can be an RNA or a DNA sequence.

In certain embodiments, use is made of chemically modified guide RNAs.Examples of guide RNA chemical modifications include, withoutlimitation, incorporation of 2′-O-methyl (M), 2′-O-methyl3′phosphorothioate (MS), or 2′-O-methyl 3′thioPACE (MSP) at one or moreterminal nucleotides. Such chemically modified guide RNAs can compriseincreased stability and increased activity as compared to unmodifiedguide RNAs, though on-target vs. off-target specificity is notpredictable. (See, Hendel, 2015, Nat Biotechnol. 33(9):985-9, doi:10.1038/nbt.3290, published online 29 Jun. 2015). Chemically modifiedguide RNAs further include, without limitation, RNAs withphosphorothioate linkages and locked nucleic acid (LNA) nucleotidescomprising a methylene bridge between the 2′ and 4′ carbons of theribose ring.

In some embodiments, the degree of complementarity, when optimallyaligned using a suitable alignment algorithm, is about or more thanabout 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimalalignment may be determined with the use of any suitable algorithm foraligning sequences, non-limiting example of which include theSmith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithmsbased on the Burrows-Wheeler Transform (e.g., the Burrows WheelerAligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies;available at www.novocraft.com), ELAND (Illumina, San Diego, Calif.),SOAP (available at soap.genomics.org.cn), and Maq (available atmaq.sourceforge.net). The ability of a guide sequence (within a nucleicacid-targeting guide RNA) to direct sequence-specific binding of anucleic acid-targeting complex to a target nucleic acid sequence may beassessed by any suitable assay. For example, the components of a nucleicacid-targeting CRISPR system sufficient to form a nucleic acid-targetingcomplex, including the guide sequence to be tested, may be provided to ahost cell having the corresponding target nucleic acid sequence, such asby transfection with vectors encoding the components of the nucleicacid-targeting complex, followed by an assessment of preferentialtargeting (e.g., cleavage) within the target nucleic acid sequence, suchas by Surveyor assay as described herein. Similarly, cleavage of atarget nucleic acid sequence may be evaluated in a test tube byproviding the target nucleic acid sequence, components of a nucleicacid-targeting complex, including the guide sequence to be tested and acontrol guide sequence different from the test guide sequence, andcomparing binding or rate of cleavage at the target sequence between thetest and control guide sequence reactions. Other assays are possible,and will occur to those skilled in the art. A guide sequence, and hencea nucleic acid-targeting guide RNA may be selected to target any targetnucleic acid sequence. The target sequence may be DNA. The targetsequence may be any RNA sequence. In some embodiments, the targetsequence may be a sequence within a RNA molecule selected from the groupconsisting of messenger RNA (mRNA), pre-mRNA, ribosomal RNA (rRNA),transfer RNA (tRNA), micro-RNA (miRNA), small interfering RNA (siRNA),small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), double strandedRNA (dsRNA), non coding RNA (ncRNA), long non-coding RNA (lncRNA), andsmall cytoplasmatic RNA (scRNA). In some preferred embodiments, thetarget sequence may be a sequence within a RNA molecule selected fromthe group consisting of mRNA, pre-mRNA, and rRNA. In some preferredembodiments, the target sequence may be a sequence within a RNA moleculeselected from the group consisting of ncRNA, and lncRNA. In some morepreferred embodiments, the target sequence may be a sequence within anmRNA molecule or a pre-mRNA molecule.

In some embodiments, a nucleic acid-targeting guide RNA is selected toreduce the degree secondary structure within the RNA-targeting guideRNA. In some embodiments, about or less than about 75%, 50%, 40%, 30%,25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the nucleicacid-targeting guide RNA participate in self-complementary base pairingwhen optimally folded. Optimal folding may be determined by any suitablepolynucleotide folding algorithm. Some programs are based on calculatingthe minimal Gibbs free energy. An example of one such algorithm ismFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981),133-148). Another example folding algorithm is the online webserverRNAfold, developed at Institute for Theoretical Chemistry at theUniversity of Vienna, using the centroid structure prediction algorithm(see e.g., A. R. Gruber et al., 2008, Cell 106(1): 23-24; and P A Carrand G M Church, 2009, Nature Biotechnology 27(12): 1151-62).

In certain embodiments, a guide RNA or crRNA may comprise, consistessentially of, or consist of a direct repeat (DR) sequence and a guidesequence or spacer sequence. In certain embodiments, the guide RNA orcrRNA may comprise, consist essentially of, or consist of a directrepeat sequence fused or linked to a guide sequence or spacer sequence.In certain embodiments, the direct repeat sequence may be locatedupstream (i.e., 5′) from the guide sequence or spacer sequence. In otherembodiments, the direct repeat sequence may be located downstream (i.e.,3′) from the guide sequence or spacer sequence.

In certain embodiments, the crRNA comprises a stem loop, preferably asingle stem loop. In certain embodiments, the direct repeat sequenceforms a stem loop, preferably a single stem loop.

In certain embodiments, the spacer length of the guide RNA is from 15 to35 nt. In certain embodiments, the spacer length of the guide RNA is atleast 15 nucleotides, preferably at least 18 nt, such at at least 19,20, 21, 22, or more nt. In certain embodiments, the spacer length isfrom 15 to 17 nt, e.g., 15, 16, or 17 nt, from 17 to 20 nt, e.g., 17,18, 19, or 20 nt, from 20 to 24 nt, e.g., 20, 21, 22, 23, or 24 nt, from23 to 25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt, e.g., 24, 25, 26,or 27 nt, from 27-30 nt, e.g., 27, 28, 29, or 30 nt, from 30-35 nt,e.g., 30, 31, 32, 33, 34, or 35 nt, or 35 nt or longer.

For minimization of toxicity and off-target effects, it will beimportant to control the concentration of RNA-targeting guide RNAdelivered. Optimal concentrations of nucleic acid-targeting guide RNAcan be determined by testing different concentrations in a cellular ornon-human eukaryote animal model and using deep sequencing the analyzethe extent of modification at potential off-target genomic loci. Theconcentration that gives the highest level of on-target modificationwhile minimizing the level of off-target modification should be chosenfor in vivo delivery. The RNA-targeting system is derived advantageouslyfrom a CRISPR-Cas13b system. In some embodiments, one or more elementsof a RNA-targeting system is derived from a particular organismcomprising an endogenous RNA-targeting system of a Cas13b effectorprotein system as herein-discussed.

The terms “orthologue” (also referred to as “ortholog” herein) and“homologue” (also referred to as “homolog” herein) are well known in theart. By means of further guidance, a “homologue” of a protein as usedherein is a protein of the same species which performs the same or asimilar function as the protein it is a homologue of Homologous proteinsmay but need not be structurally related, or are only partiallystructurally related. An “orthologue” of a protein as used herein is aprotein of a different species which performs the same or a similarfunction as the protein it is an orthologue of Orthologous proteins maybut need not be structurally related, or are only partially structurallyrelated. In particular embodiments, the homologue or orthologue of aCas13b protein as referred to herein has a sequence homology or identityof at least 50%, at least 60%, at least 70%, at least 80%, morepreferably at least 85%, even more preferably at least 90%, such as forinstance at least 95% with a Cas13b effector protein set forth in FIG.1.

It will be appreciated that any of the functionalities described hereinmay be engineered into CRISPR enzymes from other orthologs, includingchimeric enzymes comprising fragments from multiple orthologs. Examplesof such orthologs are described elsewhere herein. Thus, chimeric enzymesmay comprise fragments of CRISPR enzyme orthologs of an organism whichincludes but is not limited to Bergeyella, Prevotella, Porphyromonas,Bacteroides, Alistipes, Riemerella, Myroides, Flavobacterium,Capnocytophaga, Chryseobacterium, Phaeodactylibacter, Paludibacter orPsychroflexus. A chimeric enzyme can comprise a first fragment and asecond fragment, and the fragments, wherein one of the first and secondfragment is of or from a Cas13b effector protein of a first species (forexample, a Cas13b effector protein as listed in FIG. 1) and the otherfragment is of or from a CRISPR enzyme ortholog of a different species.

In an embodiment of the invention, there is provided an effector proteinwhich comprises an amino acid sequence which is at least 50%, 60%, 70%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or morehomologous or identical to a wild type Cas13b effector protein selectedfrom the group consisting of Porphyromonas gulae Cas13b (accessionnumber WP_039434803), Prevotella sp. P5-125 Cas13b (accession numberWP_044065294), Porphyromonas gingivalis Cas13b (accession numberWP_053444417), Porphyromonas sp. COT-052 OH4946 Cas13b (accession numberWP_039428968), Bacteroides pyogenes Cas13b (accession numberWP_034542281), Riemerella anatipestifer Cas13b (accession numberWP_004919755). The most preferred effector proteins are those at least50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more homologous or identical to a wild type Cas13b effectorprotein selected from the group consisting of Porphyromonas gulae Cas13b(accession number WP_039434803), Prevotella sp. P5-125 Cas13b (accessionnumber WP_044065294), Porphyromonas gingivalis Cas13b (accession numberWP_053444417), Porphyromonas sp. COT-052 OH4946 Cas13b (accession numberWP_039428968); and most specifically preferred are Porphyromonas gulaeCas13b (accession number WP_039434803) or Prevotella sp. P5-125 Cas13b(accession number WP_044065294).

It has been found that a number of Cas13b orthologs are characterized bycommon motifs. Accordingly, in particular embodiments, the Cas13beffector protein is a protein comprising a sequence having at least 70%sequence identity with one or more of the sequences consisting ofDKHXFGAFLNLARHN (SEQ ID NO: 118), GLLFFVSLFLDK (SEQ ID NO: 119), SKIXGFK(SEQ ID NO: 120), DMLNELXRCP (SEQ ID NO: 121), RXZDRFPYFALRYXD (SEQ IDNO: 122) and LRFQVBLGXY (SEQ ID NO: 123). In further particularembodiments, the Cas13b effector protein comprises a sequence having atleast 70% seqeuence identity at least 2, 3, 4, 5 or all 6 of thesesequences. In further particular embodiments, the sequence identity withthese sequences is at least 75%, 80%, 85%, 90%, 95% or 100%. In furtherparticular embodiments, the Cas13b effector protein is a proteincomprising a sequence having 100% sequence identity with GLLFFVSLFL (SEQID NO: 124) and RHQXRFPYF (SEQ ID NO: 125). In further particularembodiments, the Cas13b effector is a Cas13b effector protein comprisinga sequence having 100% sequence identity with RHQDRFPY (SEQ ID NO: 126).

In an embodiment of the invention, the effector protein comprises anamino acid sequence having at least 80% 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence homology or identity to a TypeVI-B effector protein consensus sequence including but not limited to aconsensus sequence described herein.

In an embodiment of the invention, the effector protein comprises atleast one HEPN domain, including but not limited to HEPN domainsdescribed herein, HEPN domains known in the art, and domains recognizedto be HEPN domains by comparison to consensus sequences and motifs. Inone non-limiting example, a consensus sequence can be derived from thesequences of Cas13b orthologs provided herein.

In an embodiment of the invention, the effector protein comprises one ormore HEPN domains comprising a RxxxxH motif sequence. The RxxxxH motifsequence can be, without limitation, from an HEPN domain describedherein or an HEPN domain known in the art. RxxxxH motifs sequencesfurther include motif sequences created by combining portions of two ormore HEPN domains.

In some embodiments, the effector protein comprises two HEPN domains. Insome embodiments, the effector protein comprises at least onecatalytically active HEPN domain comprising an RxxxxH motif. In someembodiments, the effector protein comprises two catalytically activeHEPN domains each comprising an RxxxxH motif. In some embodiments, theeffector protein comprises at least one catalytically inactive HEPNdomain obtained from mutating at least one of R or H of a wild-typeRxxxxH motif. In some embodiments, the effector protein comprises twocatalytically inactive HEPN domains each obtained from mutating at leastone of R or H of a wild-type RxxxxH motif.

In an embodiment, nucleic acid molecule(s) encoding the Type VI-BRNA-targeting effector protein may be codon-optimized for expression inan eukaryotic cell. A eukaryote can be as herein discussed. Nucleic acidmolecule(s) can be engineered or non-naturally occurring.

In an embodiment, the Type VI-B RNA-targeting effector protein, inparticular Cas13b or an ortholog or homolog thereof, may comprise one ormore mutations (and hence nucleic acid molecule(s) coding for same mayhave mutation(s)). The mutations may be artificially introducedmutations and may include but are not limited to one or more mutationsin a catalytic domain. Examples of catalytic domains with reference to aCas9 enzyme may include but are not limited to RuvC I, RuvC II, RuvC IIIand HNH domains. Examples of catalytic domains with reference to aCas13b enzyme may include but are not limited to HEPN domains.

In an embodiment, the Type VI-B protein such as Cas13b or an ortholog orhomolog thereof, may comprise one or more mutations. The mutations maybe artificially introduced mutations and may include but are not limitedto one or more mutations in a catalytic domain. Examples of catalyticdomains with reference to a Cas enzyme may include but are not limitedto HEPN domains.

In an embodiment, the Type VI-B protein such as Cas13b or an ortholog orhomolog thereof, may be used as a generic nucleic acid binding proteinwith fusion to or being operably linked to a functional domain.Exemplary functional domains may include but are not limited totranslational initiator, translational activator, translationalrepressor, nucleases, in particular ribonucleases, a spliceosome, beads,a light inducible/controllable domain or a chemicallyinducible/controllable domain.

In some embodiments, the unmodified nucleic acid-targeting effectorprotein may have cleavage activity. In some embodiments, theRNA-targeting effector protein may direct cleavage of one or bothnucleic acid (DNA or RNA) strands at the location of or near a targetsequence, such as within the target sequence and/or within thecomplement of the target sequence or at sequences associated with thetarget sequence. In some embodiments, the nucleic acid-targeting Casprotein may direct cleavage of one or both DNA or RNA strands withinabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, ormore base pairs from the first or last nucleotide of a target sequence.In some embodiments, a vector encodes a nucleic acid-targeting Casprotein that may be mutated with respect to a corresponding wild-typeenzyme such that the mutated nucleic acid-targeting Cas protein lacksthe ability to cleave RNA strands of a target polynucleotide containinga target sequence. As a further example, two or more catalytic domainsof Cas (e.g. HEPN domain) may be mutated to produce a mutated Cassubstantially lacking all RNA cleavage activity. In some embodiments, anucleic acid-targeting effector protein may be considered tosubstantially lack all RNA cleavage activity when the RNA cleavageactivity of the mutated enzyme is about no more than 25%, 10%, 5%, 1%,0.1%, 0.01%, or less of the nucleic acid cleavage activity of thenon-mutated form of the enzyme; an example can be when the nucleic acidcleavage activity of the mutated form is nil or negligible as comparedwith the non-mutated form. An effector protein may be identified withreference to the general class of enzymes that share homology to thebiggest nuclease with multiple nuclease domains from the Type VI-BCRISPR system. By derived, Applicants mean that the derived enzyme islargely based on, in the sense of having a high degree of sequencehomology with, a wildtype enzyme, but that it has been mutated(modified) in some way as known in the art or as described herein.

Again, it will be appreciated that the terms Cas and CRISPR enzyme andCRISPR protein and Cas protein are generally used interchangeably and atall points of reference herein refer by analogy to novel CRISPR effectorproteins further described in this application, unless otherwiseapparent, such as by specific reference to Cas9. As mentioned above,many of the residue numberings used herein refer to the effector proteinfrom the Type VI CRISPR locus. However, it will be appreciated that thisinvention includes many more effector proteins from other species ofmicrobes. In certain embodiments, Cas may be constitutively present orinducibly present or conditionally present or administered or delivered.Cas optimization may be used to enhance function or to develop newfunctions, one can generate chimeric Cas proteins. And Cas may be usedas a generic nucleic acid binding protein.

In some embodiments, one or more vectors driving expression of one ormore elements of a nucleic acid-targeting system are introduced into ahost cell such that expression of the elements of the nucleicacid-targeting system direct formation of a nucleic acid-targetingcomplex at one or more target sites. For example, a nucleicacid-targeting effector enzyme and a nucleic acid-targeting guide RNAcould each be operably linked to separate regulatory elements onseparate vectors. RNA(s) of the nucleic acid-targeting system can bedelivered to a transgenic nucleic acid-targeting effector protein animalor mammal, e.g., an animal or mammal that constitutively or inducibly orconditionally expresses nucleic acid-targeting effector protein; or ananimal or mammal that is otherwise expressing nucleic acid-targetingeffector proteinor has cells containing nucleic acid-targeting effectorprotein, such as by way of prior administration thereto of a vector orvectors that code for and express in vivo nucleic acid-targetingeffector protein. Alternatively, two or more of the elements expressedfrom the same or different regulatory elements, may be combined in asingle vector, with one or more additional vectors providing anycomponents of the nucleic acid-targeting system not included in thefirst vector. nucleic acid-targeting system elements that are combinedin a single vector may be arranged in any suitable orientation, such asone element located 5′ with respect to (“upstream” of) or 3′ withrespect to (“downstream” of) a second element. The coding sequence ofone element may be located on the same or opposite strand of the codingsequence of a second element, and oriented in the same or oppositedirection. In some embodiments, a single promoter drives expression of atranscript encoding a nucleic acid-targeting effector protein and thenucleic acid-targeting guide RNA, embedded within one or more intronsequences (e.g., each in a different intron, two or more in at least oneintron, or all in a single intron). In some embodiments, the nucleicacid-targeting effector protein and the nucleic acid-targeting guide RNAmay be operably linked to and expressed from the same promoter. Deliveryvehicles, vectors, particles, nanoparticles, formulations and componentsthereof for expression of one or more elements of a nucleicacid-targeting system are as used in the foregoing documents, such as WO2014/093622 (PCT/US2013/074667). In some embodiments, a vector comprisesone or more insertion sites, such as a restriction endonucleaserecognition sequence (also referred to as a “cloning site”). In someembodiments, one or more insertion sites (e.g., about or more than about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are locatedupstream and/or downstream of one or more sequence elements of one ormore vectors. In some embodiments, a vector comprises two or moreinsertion sites, so as to allow insertion of a guide sequence at eachsite. In such an arrangement, the two or more guide sequences maycomprise two or more copies of a single guide sequence, two or moredifferent guide sequences, or combinations of these. When multipledifferent guide sequences are used, a single expression construct may beused to target nucleic acid-targeting activity to multiple different,corresponding target sequences within a cell. For example, a singlevector may comprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, or more guide sequences. In some embodiments, about or morethan about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more suchguide-sequence-containing vectors may be provided, and optionallydelivered to a cell. In some embodiments, a vector comprises aregulatory element operably linked to an enzyme-coding sequence encodinga a nucleic acid-targeting effector protein. nucleic acid-targetingeffector protein or nucleic acid-targeting guide RNA or RNA(s) can bedelivered separately; and advantageously at least one of these isdelivered via a particle or nanoparticle complex. nucleic acid-targetingeffector protein mRNA can be delivered prior to the nucleicacid-targeting guide RNA to give time for nucleic acid-targetingeffector protein to be expressed. nucleic acid-targeting effectorprotein mRNA might be administered 1-12 hours (preferably around 2-6hours) prior to the administration of nucleic acid-targeting guide RNA.Alternatively, nucleic acid-targeting effector protein mRNA and nucleicacid-targeting guide RNA can be administered together. Advantageously, asecond booster dose of guide RNA can be administered 1-12 hours(preferably around 2-6 hours) after the initial administration ofnucleic acid-targeting effector protein mRNA+guide RNA. Additionaladministrations of nucleic acid-targeting effector protein mRNA and/orguide RNA might be useful to achieve the most efficient levels of genomeand/or transcriptome modification.

In one aspect, the invention provides methods for using one or moreelements of a nucleic acid-targeting system. The nucleic acid-targetingcomplex of the invention provides an effective means for modifying atarget RNA. The nucleic acid-targeting complex of the invention has awide variety of utility including modifying (e.g., deleting, inserting,translocating, inactivating, activating) a target RNA in a multiplicityof cell types. As such the nucleic acid-targeting complex of theinvention has a broad spectrum of applications in, e.g., gene therapy,drug screening, disease diagnosis, and prognosis. An exemplary nucleicacid-targeting complex comprises a RNA-targeting effector proteincomplexed with a guide RNA hybridized to a target sequence within thetarget locus of interest.

In one embodiment, this invention provides a method of cleaving a targetRNA. The method may comprise modifying a target RNA using a nucleicacid-targeting complex that binds to the target RNA and effect cleavageof said target RNA. In an embodiment, the nucleic acid-targeting complexof the invention, when introduced into a cell, may create a break (e.g.,a single or a double strand break) in the RNA sequence. For example, themethod can be used to cleave a disease RNA in a cell. For example, anexogenous RNA template comprising a sequence to be integrated flanked byan upstream sequence and a downstream sequence may be introduced into acell. The upstream and downstream sequences share sequence similaritywith either side of the site of integration in the RNA. Where desired, adonor RNA can be mRNA. The exogenous RNA template comprises a sequenceto be integrated (e.g., a mutated RNA). The sequence for integration maybe a sequence endogenous or exogenous to the cell. Examples of asequence to be integrated include RNA encoding a protein or a non-codingRNA (e.g., a microRNA). Thus, the sequence for integration may beoperably linked to an appropriate control sequence or sequences.Alternatively, the sequence to be integrated may provide a regulatoryfunction. The upstream and downstream sequences in the exogenous RNAtemplate are selected to promote recombination between the RNA sequenceof interest and the donor RNA. The upstream sequence is a RNA sequencethat shares sequence similarity with the RNA sequence upstream of thetargeted site for integration. Similarly, the downstream sequence is aRNA sequence that shares sequence similarity with the RNA sequencedownstream of the targeted site of integration. The upstream anddownstream sequences in the exogenous RNA template can have 75%, 80%,85%, 90%, 95%, or 100% sequence identity with the targeted RNA sequence.Preferably, the upstream and downstream sequences in the exogenous RNAtemplate have about 95%, 96%, 97%, 98%, 99%, or 100% sequence identitywith the targeted RNA sequence. In some methods, the upstream anddownstream sequences in the exogenous RNA template have about 99% or100% sequence identity with the targeted RNA sequence. An upstream ordownstream sequence may comprise from about 20 bp to about 2500 bp, forexample, about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200,2300, 2400, or 2500 bp. In some methods, the exemplary upstream ordownstream sequence have about 200 bp to about 2000 bp, about 600 bp toabout 1000 bp, or more particularly about 700 bp to about 1000 bp. Insome methods, the exogenous RNA template may further comprise a marker.Such a marker may make it easy to screen for targeted integrations.Examples of suitable markers include restriction sites, fluorescentproteins, or selectable markers. The exogenous RNA template of theinvention can be constructed using recombinant techniques (see, forexample, Sambrook et al., 2001 and Ausubel et al., 1996). In a methodfor modifying a target RNA by integrating an exogenous RNA template, abreak (e.g., double or single stranded break in double or singlestranded DNA or RNA) is introduced into the DNA or RNA sequence by thenucleic acid-targeting complex, the break is repaired via homologousrecombination with an exogenous RNA template such that the template isintegrated into the RNA target. The presence of a double-stranded breakfacilitates integration of the template. In other embodiments, thisinvention provides a method of modifying expression of a RNA in aeukaryotic cell. The method comprises increasing or decreasingexpression of a target polynucleotide by using a nucleic acid-targetingcomplex that binds to the RNA (e.g., mRNA or pre-mRNA). In some methods,a target RNA can be inactivated to effect the modification of theexpression in a cell. For example, upon the binding of a RNA-targetingcomplex to a target sequence in a cell, the target RNA is inactivatedsuch that the sequence is not translated, the coded protein is notproduced, or the sequence does not function as the wild-type sequencedoes. For example, a protein or microRNA coding sequence may beinactivated such that the protein or microRNA or pre-microRNA transcriptis not produced. The target RNA of a RNA-targeting complex can be anyRNA endogenous or exogenous to the eukaryotic cell. For example, thetarget RNA can be a RNA residing in the nucleus of the eukaryotic cell.The target RNA can be a sequence (e.g., mRNA or pre-mRNA) coding a geneproduct (e.g., a protein) or a non-coding sequence (e.g., ncRNA, lncRNA,tRNA, or rRNA). Examples of target RNA include a sequence associatedwith a signaling biochemical pathway, e.g., a signaling biochemicalpathway-associated RNA. Examples of target RNA include a diseaseassociated RNA. A “disease-associated” RNA refers to any RNA which isyielding translation products at an abnormal level or in an abnormalform in cells derived from a disease-affected tissues compared withtissues or cells of a non disease control. It may be a RNA transcribedfrom a gene that becomes expressed at an abnormally high level; it maybe a RNA transcribed from a gene that becomes expressed at an abnormallylow level, where the altered expression correlates with the occurrenceand/or progression of the disease. A disease-associated RNA also refersto a RNA transcribed from a gene possessing mutation(s) or geneticvariation that is directly responsible or is in linkage disequilibriumwith a gene(s) that is responsible for the etiology of a disease. Thetranslated products may be known or unknown, and may be at a normal orabnormal level. The target RNA of a RNA-targeting complex can be any RNAendogenous or exogenous to the eukaryotic cell. For example, the targetRNA can be a RNA residing in the nucleus of the eukaryotic cell. Thetarget RNA can be a sequence (e.g., mRNA or pre-mRNA) coding a geneproduct (e.g., a protein) or a non-coding sequence (e.g., ncRNA, lncRNA,tRNA, or rRNA).

In some embodiments, the method may comprise allowing a nucleicacid-targeting complex to bind to the target RNA to effect cleavage ofsaid target RNA or RNA thereby modifying the target RNA, wherein thenucleic acid-targeting complex comprises a nucleic acid-targetingeffector protein complexed with a guide RNA hybridized to a targetsequence within said target RNA. In one aspect, the invention provides amethod of modifying expression of RNA in a eukaryotic cell. In someembodiments, the method comprises allowing a nucleic acid-targetingcomplex to bind to the RNA such that said binding results in increasedor decreased expression of said RNA; wherein the nucleic acid-targetingcomplex comprises a nucleic acid-targeting effector protein complexedwith a guide RNA. Similar considerations and conditions apply as abovefor methods of modifying a target RNA. In fact, these sampling,culturing and re-introduction options apply across the aspects of thepresent invention. In one aspect, the invention provides for methods ofmodifying a target RNA in a eukaryotic cell, which may be in vivo, exvivo or in vitro. In some embodiments, the method comprises sampling acell or population of cells from a human or non-human animal, andmodifying the cell or cells. Culturing may occur at any stage ex vivo.The cell or cells may even be re-introduced into the non-human animal orplant. For re-introduced cells it is particularly preferred that thecells are stem cells.

Indeed, in any aspect of the invention, the nucleic acid-targetingcomplex may comprise a nucleic acid-targeting effector protein complexedwith a guide RNA hybridized to a target sequence.

The invention relates to the engineering and optimization of systems,methods and compositions used for the control of gene expressioninvolving RNA sequence targeting, that relate to the nucleicacid-targeting system and components thereof. In advantageousembodiments, the effector protein is a Type VI-B protein such as Cas13b.An advantage of the present methods is that the CRISPR system minimizesor avoids off-target binding and its resulting side effects. This isachieved using systems arranged to have a high degree of sequencespecificity for the target RNA.

The use of two different aptamers (each associated with a distinctnucleic acid-targeting guide RNAs) allows an activator-adaptor proteinfusion and a repressor-adaptor protein fusion to be used, with differentnucleic acid-targeting guide RNAs, to activate expression of one DNA orRNA, whilst repressing another. They, along with their different guideRNAs can be administered together, or substantially together, in amultiplexed approach. A large number of such modified nucleicacid-targeting guide RNAs can be used all at the same time, for example10 or 20 or 30 and so forth, whilst only one (or at least a minimalnumber) of effector protein molecules need to be delivered, as acomparatively small number of effector protein molecules can be usedwith a large number modified guides. The adaptor protein may beassociated (preferably linked or fused to) one or more activators or oneor more repressors. For example, the adaptor protein may be associatedwith a first activator and a second activator. The first and secondactivators may be the same, but they are preferably differentactivators. Three or more or even four or more activators (orrepressors) may be used, but package size may limit the number beinghigher than 5 different functional domains. Linkers are preferably used,over a direct fusion to the adaptor protein, where two or morefunctional domains are associated with the adaptor protein. Suitablelinkers might include the GlySer linker.

It is also envisaged that the nucleic acid-targeting effectorprotein-guide RNA complex as a whole may be associated with two or morefunctional domains. For example, there may be two or more functionaldomains associated with the nucleic acid-targeting effector protein, orthere may be two or more functional domains associated with the guideRNA (via one or more adaptor proteins), or there may be one or morefunctional domains associated with the nucleic acid-targeting effectorprotein and one or more functional domains associated with the guide RNA(via one or more adaptor proteins).

The fusion between the adaptor protein and the activator or repressormay include a linker. For example, GlySer linkers GGGS (SEQ ID NO: 127)can be used. They can be used in repeats of 3 ((GGGGS)₃) (SEQ ID NO:128) or 6 (SEQ ID NO: 129), 9 (SEQ ID NO: 130) or even 12 (SEQ ID NO:131) or more, to provide suitable lengths, as required. Linkers can beused between the guide RNAs and the functional domain (activator orrepressor), or between the nucleic acid-targeting effector protein andthe functional domain (activator or repressor). The linkers the user toengineer appropriate amounts of “mechanical flexibility”.

The invention comprehends a nucleic acid-targeting complex comprising anucleic acid-targeting effector protein and a guide RNA, wherein thenucleic acid-targeting effector protein comprises at least one mutation,such that the nucleic acid-targeting Cas protein has no more than 5% ofthe activity of the nucleic acid-targeting Cas protein not having the atleast one mutation and, optionally, at least one or more nuclearlocalization sequences; the guide RNA comprises a guide sequence capableof hybridizing to a target sequence in a RNA of interest in a cell; andwherein: the nucleic acid-targeting effector protein is associated withtwo or more functional domains; or at least one loop of the guide RNA ismodified by the insertion of distinct RNA sequence(s) that bind to oneor more adaptor proteins, and wherein the adaptor protein is associatedwith two or more functional domains; or the nucleic acid-targetingeffector protein is associated with one or more functional domains andat least one loop of the guide RNA is modified by the insertion ofdistinct RNA sequence(s) that bind to one or more adaptor proteins, andwherein the adaptor protein is associated with one or more functionaldomains.

Cas13b Effector Protein Complexes can Deliver Functional Effectors

Unlike CRISPR-Cas13b-mediated knockout, which eliminates expression bymutating at the RNA level, CRISPR-Cas13b knockdown allows for temporaryreduction of gene expression through the use of artificial transcriptionfactors, e.g., via mutating residues in cleavage domain(s) of the Cas13bprotein results in the generation of a catalytically inactive Cas13bprotein. A catalytically inactive Cas13b complexes with a guide RNA orcrRNA and localizes to the RNA sequence specified by that guide RNA's orcrRNA's targeting domain, however, it does not cleave the target. Fusionof the inactive Cas13b protein to an effector domain, e.g., atranscription repression domain, enables recruitment of the effector toany site specified by the guide RNA.

Optimized Functional RNA Targeting Systems

In an aspect the invention thus provides a system for specific deliveryof functional components to the RNA environment. This can be ensuredusing the CRISPR systems comprising the RNA targeting effector proteinsof the present invention which allow specific targeting of differentcomponents to RNA. More particularly such components include activatorsor repressors, such as activators or repressors of RNA translation,degradation, etc.

According to one aspect the invention provides non-naturally occurringor engineered composition comprising a guide RNA or crRNA comprising aguide sequence capable of hybridizing to a target sequence of interestin a cell, wherein the guide RNA or crRNA is modified by the insertionof one or more distinct RNA sequence(s) that bind an adaptor protein. Inparticular embodiments, the RNA sequences may bind to two or moreadaptor proteins (e.g. aptamers), and wherein each adaptor protein isassociated with one or more functional domains. When there is more thanone functional domain, the functional domains can be same or different,e.g., two of the same or two different activators or repressors. In anaspect the invention provides a herein-discussed composition, whereinthe one or more functional domains are attached to the RNA targetingenzyme so that upon binding to the target RNA the functional domain isin a spatial orientation allowing for the functional domain to functionin its attributed function; In an aspect the invention provides aherein-discussed composition, wherein the composition comprises aCRISPR-Cas13b complex having at least three functional domains, at leastone of which is associated with the RNA targeting enzyme and at leasttwo of which are associated with the gRNA or crRNA.

Delivery of the Cas13b Effector Protein Complex or Components Thereof

Through this disclosure and the knowledge in the art, TALEs, CRISPR-Cassystems, or components thereof or nucleic acid molecules thereof(including, for instance HDR template) or nucleic acid moleculesencoding or providing components thereof may be delivered by a deliverysystem herein described both generally and in detail.

Vector delivery, e.g., plasmid, viral delivery: The CRISPR enzyme,and/or any of the present RNAs, for instance a guide RNA, can bedelivered using any suitable vector, e.g., plasmid or viral vectors,such as adeno associated virus (AAV), lentivirus, adenovirus or otherviral vector types, or combinations thereof. Effector proteins and oneor more guide RNAs can be packaged into one or more vectors, e.g.,plasmid or viral vectors. In some embodiments, the vector, e.g., plasmidor viral vector is delivered to the tissue of interest by, for example,an intramuscular injection, while other times the delivery is viaintravenous, transdermal, intranasal, oral, mucosal, or other deliverymethods. Such delivery may be either via a single dose, or multipledoses. One skilled in the art understands that the actual dosage to bedelivered herein may vary greatly depending upon a variety of factors,such as the vector choice, the target cell, organism, or tissue, thegeneral condition of the subject to be treated, the degree oftransformation/modification sought, the administration route, theadministration mode, the type of transformation/modification sought,etc.

Such a dosage may further contain, for example, a carrier (water,saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin,dextran, agar, pectin, peanut oil, sesame oil, etc.), a diluent, apharmaceutically-acceptable carrier (e.g., phosphate-buffered saline), apharmaceutically-acceptable excipient, and/or other compounds known inthe art. The dosage may further contain one or more pharmaceuticallyacceptable salts such as, for example, a mineral acid salt such as ahydrochloride, a hydrobromide, a phosphate, a sulfate, etc.; and thesalts of organic acids such as acetates, propionates, malonates,benzoates, etc. Additionally, auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, gels or gelling materials,flavorings, colorants, microspheres, polymers, suspension agents, etc.may also be present herein. In addition, one or more other conventionalpharmaceutical ingredients, such as preservatives, humectants,suspending agents, surfactants, antioxidants, anticaking agents,fillers, chelating agents, coating agents, chemical stabilizers, etc.may also be present, especially if the dosage form is a reconstitutableform. Suitable exemplary ingredients include microcrystalline cellulose,carboxymethylcellulose sodium, polysorbate 80, phenylethyl alcohol,chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propylgallate, the parabens, ethyl vanillin, glycerin, phenol,parachlorophenol, gelatin, albumin and a combination thereof. A thoroughdiscussion of pharmaceutically acceptable excipients is available inREMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991) which isincorporated by reference herein.

In an embodiment herein the delivery is via an adenovirus, which may beat a single booster dose containing at least 1×10⁵ particles (alsoreferred to as particle units, pu) of adenoviral vector. In anembodiment herein, the dose preferably is at least about 1×10⁶ particles(for example, about 1×10⁶-1×10¹² particles), more preferably at leastabout 1×10′ particles, more preferably at least about 1×10⁸ particles(e.g., about 1×10⁸-1×10¹¹ particles or about 1×10⁸-1×10¹² particles),and most preferably at least about 1×10° particles (e.g., about1×10⁹-1×10¹⁰ particles or about 1×10⁹-1×10¹² particles), or even atleast about 1×10¹⁰ particles (e.g., about 1×10¹⁰-1×10¹² particles) ofthe adenoviral vector. Alternatively, the dose comprises no more thanabout 1×10¹⁴ particles, preferably no more than about 1×10¹³ particles,even more preferably no more than about 1×10¹² particles, even morepreferably no more than about 1×10¹¹ particles, and most preferably nomore than about 1×10¹⁰ particles (e.g., no more than about 1×10⁹articles). Thus, the dose may contain a single dose of adenoviral vectorwith, for example, about 1×10⁶ particle units (pu), about 2×10⁶ pu,about 4×10⁶ pu, about 1×10′ pu, about 2×10′ pu, about 4×10′ pu, about1×10⁸ pu, about 2×10⁸ pu, about 4×10⁸ pu, about 1×10⁹ pu, about 2×10⁹pu, about 4×10⁹ pu, about 1×10¹⁰ pu, about 2×10¹⁰ pu, about 4×10¹⁰ pu,about 1×10¹¹ pu, about 2×10¹¹ pu, about 4×10¹¹ pu, about 1×10¹² pu,about 2×10¹² pu, or about 4×10¹² pu of adenoviral vector. See, forexample, the adenoviral vectors in U.S. Pat. No. 8,454,972 B2 to Nabel,et. al., granted on Jun. 4, 2013; incorporated by reference herein, andthe dosages at col 29, lines 36-58 thereof. In an embodiment herein, theadenovirus is delivered via multiple doses.

In an embodiment herein, the delivery is via an AAV. A therapeuticallyeffective dosage for in vivo delivery of the AAV to a human is believedto be in the range of from about 20 to about 50 ml of saline solutioncontaining from about 1×10¹⁰ to about 1×10¹⁰ functional AAV/ml solution.The dosage may be adjusted to balance the therapeutic benefit againstany side effects. In an embodiment herein, the AAV dose is generally inthe range of concentrations of from about 1×10⁵ to 1×10⁵⁰ genomes AAV,from about 1×10⁸ to 1×10²⁰ genomes AAV, from about 1×10¹⁰ to about1×10¹⁶ genomes, or about 1×10¹¹ to about 1×10¹⁶ genomes AAV. A humandosage may be about 1×10¹³ genomes AAV. Such concentrations may bedelivered in from about 0.001 ml to about 100 ml, about 0.05 to about 50ml, or about 10 to about 25 ml of a carrier solution. Other effectivedosages can be readily established by one of ordinary skill in the artthrough routine trials establishing dose response curves. See, forexample, U.S. Pat. No. 8,404,658 B2 to Hajjar, et al., granted on Mar.26, 2013, at col. 27, lines 45-60.

In an embodiment herein the delivery is via a plasmid. In such plasmidcompositions, the dosage should be a sufficient amount of plasmid toelicit a response. For instance, suitable quantities of plasmid DNA inplasmid compositions can be from about 0.1 to about 2 mg, or from about1 μg to about 10 μg per 70 kg individual. Plasmids of the invention willgenerally comprise (i) a promoter; (ii) a sequence encoding an nucleicacid-targeting CRISPR enzyme, operably linked to said promoter; (iii) aselectable marker; (iv) an origin of replication; and (v) atranscription terminator downstream of and operably linked to (ii). Theplasmid can also encode the RNA components of a CRISPR complex, but oneor more of these may instead be encoded on a different vector.

The doses herein are based on an average 70 kg individual. The frequencyof administration is within the ambit of the medical or veterinarypractitioner (e.g., physician, veterinarian), or scientist skilled inthe art. It is also noted that mice used in experiments are typicallyabout 20 g and from mice experiments one can scale up to a 70 kgindividual.

In some embodiments the RNA molecules of the invention are delivered inliposome or lipofectin formulations and the like and can be prepared bymethods well known to those skilled in the art. Such methods aredescribed, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and5,580,859, which are herein incorporated by reference. Delivery systemsaimed specifically at the enhanced and improved delivery of siRNA intomammalian cells have been developed, (see, for example, Shen et al FEBSLet. 2003, 539:111-114; Xia et al., Nat. Biotech. 2002, 20:1006-1010;Reich et al., Mol. Vision. 2003, 9: 210-216; Sorensen et al., J. Mol.Biol. 2003, 327: 761-766; Lewis et al., Nat. Gen. 2002, 32: 107-108 andSimeoni et al., NAR 2003, 31, 11: 2717-2724) and may be applied to thepresent invention. siRNA has recently been successfully used forinhibition of gene expression in primates (see for example. Tolentino etal., Retina 24(4):660 which may also be applied to the presentinvention.

Indeed, RNA delivery is a useful method of in vivo delivery. It ispossible to deliver nucleic acid-targeting Cas protein and guide RNA(and, for instance, HR repair template) into cells using liposomes orparticles. Thus delivery of the nucleic acid-targeting Cas13b proteinand/or delivery of the guide RNAs or crRNAs of the invention may be inRNA form and via microvesicles, liposomes or particles. For example,Cas13b mRNA and guide RNA or crRNA can be packaged into liposomalparticles for delivery in vivo. Liposomal transfection reagents such aslipofectamine from Life Technologies and other reagents on the marketcan effectively deliver RNA molecules into the liver.

Means of delivery of RNA also preferred include delivery of RNA viananoparticles (Cho, S., Goldberg, M., Son, S., Xu, Q., Yang, F., Mei,Y., Bogatyrev, S., Langer, R. and Anderson, D., Lipid-like nanoparticlesfor small interfering RNA delivery to endothelial cells, AdvancedFunctional Materials, 19: 3112-3118, 2010) or exosomes (Schroeder, A.,Levins, C., Cortez, C., Langer, R., and Anderson, D., Lipid-basednanotherapeutics for siRNA delivery, Journal of Internal Medicine, 267:9-21, 2010, PMID: 20059641). Indeed, exosomes have been shown to beparticularly useful in delivery siRNA, a system with some parallels tothe RNA-targeting system. For instance, El-Andaloussi S, et al.(“Exosome-mediated delivery of siRNA in vitro and in vivo.” Nat Protoc.2012 December; 7(12):2112-26. doi: 10.1038/nprot.2012.131. Epub 2012Nov. 15) describe how exosomes are promising tools for drug deliveryacross different biological barriers and can be harnessed for deliveryof siRNA in vitro and in vivo. Their approach is to generate targetedexosomes through transfection of an expression vector, comprising anexosomal protein fused with a peptide ligand. The exosomes are thenpurify and characterized from transfected cell supernatant, then RNA isloaded into the exosomes. Delivery or administration according to theinvention can be performed with exosomes, in particular but not limitedto the brain. Vitamin E (α-tocopherol) may be conjugated with nucleicacid-targeting Cas protein and delivered to the brain along with highdensity lipoprotein (HDL), for example in a similar manner as was doneby Uno et al. (HUMAN GENE THERAPY 22:711-719 (June 2011)) for deliveringshort-interfering RNA (siRNA) to the brain. Mice were infused viaOsmotic minipumps (model 1007D; Alzet, Cupertino, Calif.) filled withphosphate-buffered saline (PBS) or free TocsiBACE or Toc-siBACE/HDL andconnected with Brain Infusion Kit 3 (Alzet). A brain-infusion cannulawas placed about 0.5 mm posterior to the bregma at midline for infusioninto the dorsal third ventricle. Uno et al. found that as little as 3nmol of Toc-siRNA with HDL could induce a target reduction in comparabledegree by the same ICV infusion method. A similar dosage of nucleicacid-targeting effector protein conjugated to α-tocopherol andco-administered with HDL targeted to the brain may be contemplated forhumans in the present invention, for example, about 3 nmol to about 3μmol of nucleic acid-targeting effector protein targeted to the brainmay be contemplated. Zou et al. ((HUMAN GENE THERAPY 22:465-475 (April2011)) describes a method of lentiviral-mediated delivery ofshort-hairpin RNAs targeting PKCy for in vivo gene silencing in thespinal cord of rats. Zou et al. administered about 10₁1.1 of arecombinant lentivirus having a titer of 1×10⁹ transducing units (TU)/mlby an intrathecal catheter. A similar dosage of nucleic acid-targetingeffector protein expressed in a lentiviral vector targeted to the brainmay be contemplated for humans in the present invention, for example,about 10-50 ml of nucleic acid-targeting effector protein targeted tothe brain in a lentivirus having a titer of 1×10⁹ transducing units(TU)/ml may be contemplated.

In terms of local delivery to the brain, this can be achieved in variousways. For instance, material can be delivered intrastriatally e.g., byinjection. Injection can be performed stereotactically via a craniotomy.

Packaging and Promoters Generally

Ways to package RNA-targeting effector protein (Cas13b proteins) codingnucleic acid molecules, e.g., DNA, into vectors, e.g., viral vectors, tomediate genome modification in vivo include:

-   -   Single virus vector:        -   Vector containing two or more expression cassettes:        -   Promoter-nucleic acid-targeting effector protein coding            nucleic acid molecule—terminator        -   Promoter-guide RNA1-terminator        -   Promoter-guide RNA (N)-terminator (up to size limit of            vector)    -   Double virus vector:        -   Vector 1 containing one expression cassette for driving the            expression of RNA-targeting effector protein (Cas13b)        -   Promoter-RNA-targeting effector (Cas13b) protein coding            nucleic acid molecule-terminator        -   Vector 2 containing one more expression cassettes for            driving the expression of one or more guideRNAs or crRNAs        -   Promoter-guide RNA1 or crRNA1-terminator        -   Promoter-guide RNA1 (N) or crRNA1 (N)-terminator (up to size            limit of vector).

The promoter used to drive RNA-targeting effector protein coding nucleicacid molecule expression can include AAV ITR can serve as a promoter:this is advantageous for eliminating the need for an additional promoterelement (which can take up space in the vector). The additional spacefreed up can be used to drive the expression of additional elements(gRNA, etc.). Also, ITR activity is relatively weaker, so can be used toreduce potential toxicity due to over expression of nucleicacid-targeting effector protein. For ubiquitous expression, can usepromoters: CMV, CAG, CBh, PGK, SV40, Ferritin heavy or light chains,etc. For brain or other CNS expression, can use promoters: SynapsinI forall neurons, CaMKIIalpha for excitatory neurons, GAD67 or GAD65 or VGATfor GABAergic neurons, etc. For liver expression, can use Albuminpromoter. For lung expression, can use SP-B. For endothelial cells, canuse ICAM. For hematopoietic cells can use IFNbeta or CD45. ForOsteoblasts can use OG-2. The promoter used to drive guide RNA caninclude: Pol III promoters such as U6 or H1; Pol II promoter andintronic cassettes to express guide RNA or crRNA.

Adeno Associated Virus (AAV)

Cas13b and one or more guide RNA or crRNA can be delivered using adenoassociated virus (AAV), lentivirus, adenovirus or other plasmid or viralvector types, in particular, using formulations and doses from, forexample, U.S. Pat. No. 8,454,972 (formulations, doses for adenovirus),U.S. Pat. No. 8,404,658 (formulations, doses for AAV) and U.S. Pat. No.5,846,946 (formulations, doses for DNA plasmids) and from clinicaltrials and publications regarding the clinical trials involvinglentivirus, AAV and adenovirus. For examples, for AAV, the route ofadministration, formulation and dose can be as in U.S. Pat. No.8,454,972 and as in clinical trials involving AAV. For Adenovirus, theroute of administration, formulation and dose can be as in U.S. Pat. No.8,404,658 and as in clinical trials involving adenovirus. For plasmiddelivery, the route of administration, formulation and dose can be as inU.S. Pat. No. 5,846,946 and as in clinical studies involving plasmids.Doses may be based on or extrapolated to an average 70 kg individual(e.g., a male adult human), and can be adjusted for patients, subjects,mammals of different weight and species. Frequency of administration iswithin the ambit of the medical or veterinary practitioner (e.g.,physician, veterinarian), depending on usual factors including the age,sex, general health, other conditions of the patient or subject and theparticular condition or symptoms being addressed. The viral vectors canbe injected into the tissue of interest. For cell-type specific genomemodification, the expression of RNA-targeting effector protein (Cas13beffector protein) can be driven by a cell-type specific promoter. Forexample, liver-specific expression might use the Albumin promoter andneuron-specific expression (e.g., for targeting CNS disorders) might usethe Synapsin I promoter. In terms of in vivo delivery, AAV isadvantageous over other viral vectors for a couple of reasons: Lowtoxicity (this may be due to the purification method not requiring ultracentrifugation of cell particles that can activate the immune response)and Low probability of causing insertional mutagenesis because itdoesn't integrate into the host genome.

AAV has a packaging limit of 4.5 or 4.75 Kb. This means that theRNA-targeting effector protein (Cas13b effector protein) coding sequenceas well as a promoter and transcription terminator have to be all fitinto the same viral vector. As to AAV, the AAV can be AAV1, AAV2, AAV5or any combination thereof. One can select the AAV of the AAV withregard to the cells to be targeted; e.g., one can select AAV serotypes1, 2, 5 or a hybrid capsid AAV1, AAV2, AAV5 or any combination thereoffor targeting brain or neuronal cells; and one can select AAV4 fortargeting cardiac tissue. AAV8 is useful for delivery to the liver. Theherein promoters and vectors are preferred individually. A tabulation ofcertain AAV serotypes as to these cells (see Grimm, D. et al, J. Virol.82: 5887-5911 (2008)) is as follows:

Cell Line AAV-1 AAV-2 AAV-3 AAV-4 AAV-5 AAV-6 AAV-8 AAV-9 Huh-7 13 1002.5 0.0 0.1 10 0.7 0.0 HEK293 25 100 2.5 0.1 0.1 5 0.7 0.1 HeLa 3 1002.0 0.1 6.7 1 0.2 0.1 HepG2 3 100 16.7 0.3 1.7 5 0.3 ND Hep1A 20 100 0.21.0 0.1 1 0.2 0.0 911 17 100 11 0.2 0.1 17 0.1 ND CHO 100 100 14 1.4 33350 10 1.0 COS 33 100 33 3.3 5.0 14 2.0 0.5 MeWo 10 100 20 0.3 6.7 10 1.00.2 NIH3T3 10 100 2.9 2.9 0.3 10 0.3 ND A549 14 100 20 ND 0.5 10 0.5 0.1HT1180 20 100 10 0.1 0.3 33 0.5 0.1 Monocytes 1111 100 ND ND 125 1429 NDND Immature DC 2500 100 ND ND 222 2857 ND ND Mature DC 2222 100 ND ND333 3333 ND ND

Lentivirus

Lentiviruses are complex retroviruses that have the ability to infectand express their genes in both mitotic and post-mitotic cells. The mostcommonly known lentivirus is the human immunodeficiency virus (HIV),which uses the envelope glycoproteins of other viruses to target a broadrange of cell types. Lentiviruses may be prepared as follows. Aftercloning pCasES10 (which contains a lentiviral transfer plasmidbackbone), HEK293FT at low passage (p=5) were seeded in a T-75 flask to50% confluence the day before transfection in DMEM with 10% fetal bovineserum and without antibiotics. After 20 hours, media was changed toOptiMEM (serum-free) media and transfection was done 4 hours later.Cells were transfected with 10 μg of lentiviral transfer plasmid(pCasES10) and the following packaging plasmids: 5 μg of pMD2.G (VSV-gpseudotype), and 7.5 ug of psPAX2 (gag/pol/rev/tat). Transfection wasdone in 4 mL OptiMEM with a cationic lipid delivery agent (50 uLLipofectamine 2000 and 100 ul Plus reagent). After 6 hours, the mediawas changed to antibiotic-free DMEM with 10% fetal bovine serum. Thesemethods use serum during cell culture, but serum-free methods arepreferred.

Lentivirus may be purified as follows. Viral supernatants were harvestedafter 48 hours. Supernatants were first cleared of debris and filteredthrough a 0.45 um low protein binding (PVDF) filter. They were then spunin a ultracentrifuge for 2 hours at 24,000 rpm. Viral pellets wereresuspended in 50 ul of DMEM overnight at 4C. They were then aliquottedand immediately frozen at −80° C.

In another embodiment, minimal non-primate lentiviral vectors based onthe equine infectious anemia virus (EIAV) are also contemplated,especially for ocular gene therapy (see, e.g., Balagaan, J Gene Med2006; 8: 275-285). In another embodiment, RetinoStat®, an equineinffctious anemia virus-based lentiviral gene therapy vector thatexpresses angiostatic proteins endostatin and angiostatin that isdelivered via a subretinal injection for the treatment of the web formof age-related macular degeneration is also contemplated (see, e.g.,Binley et al., HUMAN GENE THERAPY 23:980-991 (September 2012)) and thisvector may be modified for the nucleic acid-targeting system of thepresent invention.

In another embodiment, self-inactivating lentiviral vectors with ansiRNA targeting a common exon shared by HIV tat/rev, anucleolar-localizing TAR decoy, and an anti-CCR5-specific hammerheadribozyme (see, e.g., DiGiusto et al. (2010) Sci Transl Med 2:36ra43) maybe used/and or adapted to the nucleic acid-targeting system of thepresent invention. A minimum of 2.5×10⁶ CD34+cells per kilogram patientweight may be collected and prestimulated for 16 to 20 hours in X-VIVO15 medium (Lonza) containing 2 μmol/L-glutamine, stem cell factor (100ng/ml), Flt-3 ligand (Flt-3L) (100 ng/ml), and thrombopoietin (10 ng/ml)(CellGenix) at a density of 2×10⁶ cells/ml. Prestimulated cells may betransduced with lentiviral at a multiplicity of infection of 5 for 16 to24 hours in 75-cm² tissue culture flasks coated with fibronectin (25mg/cm²) (RetroNectin, Takara Bio Inc.).

Lentiviral vectors have been disclosed as in the treatment forParkinson's Disease, see, e.g., US Patent Publication No. 20120295960and U.S. Pat. Nos. 7,303,910 and 7,351,585. Lentiviral vectors have alsobeen disclosed for the treatment of ocular diseases, see e.g., US PatentPublication Nos. 20060281180, 20090007284, US20110117189; US20090017543;US20070054961, US20100317109. Lentiviral vectors have also beendisclosed for delivery to the brain, see, e.g., US Patent PublicationNos. US20110293571; US20110293571, US20040013648, US20070025970,US20090111106 and U.S. Pat. No. 7,259,015.

RNA Delivery

RNA delivery: The nucleic acid-targeting Cas13b protein, and/or guideRNA, can also be delivered in the form of RNA. mRNA can be synthesizedusing a PCR cassette containing the following elements:T7_promoter-kozak sequence (GCCACC)-effector protrein-3′ UTR from betaglobin-polyA tail (a string of 120 or more adenines). The cassette canbe used for transcription by T7 polymerase. Guide RNAs or crRNAs canalso be transcribed using in vitro transcription from a cassettecontaining T7_promoter-GG-guide RNA or crRNA sequence.

Particle Delivery Systems and/or Formulations:

Several types of particle delivery systems and/or formulations are knownto be useful in a diverse spectrum of biomedical applications. Ingeneral, a particle is defined as a small object that behaves as a wholeunit with respect to its transport and properties. Particles are furtherclassified according to diameter. Coarse particles cover a range between2,500 and 10,000 nanometers. Fine particles are sized between 100 and2,500 nanometers. Ultrafine particles, or nanoparticles, are generallybetween 1 and 100 nanometers in size. The basis of the 100-nm limit isthe fact that novel properties that differentiate particles from thebulk material typically develop at a critical length scale of under 100nm.

As used herein, a particle delivery system/formulation is defined as anybiological delivery system/formulation which includes a particle inaccordance with the present invention. A particle in accordance with thepresent invention is any entity having a greatest dimension (e.g.diameter) of less than 100 microns (μm). In some embodiments, inventiveparticles have a greatest dimension of less than 10 μm. In someembodiments, inventive particles have a greatest dimension of less than2000 nanometers (nm). In some embodiments, inventive particles have agreatest dimension of less than 1000 nanometers (nm). In someembodiments, inventive particles have a greatest dimension of less than900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100nm. Typically, inventive particles have a greatest dimension (e.g.,diameter) of 500 nm or less. In some embodiments, inventive particleshave a greatest dimension (e.g., diameter) of 250 nm or less. In someembodiments, inventive particles have a greatest dimension (e.g.,diameter) of 200 nm or less. In some embodiments, inventive particleshave a greatest dimension (e.g., diameter) of 150 nm or less. In someembodiments, inventive particles have a greatest dimension (e.g.,diameter) of 100 nm or less. Smaller particles, e.g., having a greatestdimension of 50 nm or less are used in some embodiments of theinvention. In some embodiments, inventive particles have a greatestdimension ranging between 25 nm and 200 nm.

Particle characterization (including e.g., characterizing morphology,dimension, etc.) is done using a variety of different techniques. Commontechniques are electron microscopy (TEM, SEM), atomic force microscopy(AFM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy(XPS), powder X-ray diffraction (XRD), Fourier transform infraredspectroscopy (FTIR), matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (MALDI-TOF), ultraviolet-visiblespectroscopy, dual polarisation interferometry and nuclear magneticresonance (NMR). Characterization (dimension measurements) may be madeas to native particles (i.e., preloading) or after loading of the cargo(herein cargo refers to e.g., one or more components of CRISPR-Cas13bsystem e.g., Cas13b enzyme or mRNA or guide RNA, or any combinationthereof, and may include additional carriers and/or excipients) toprovide particles of an optimal size for delivery for any in vitro, exvivo and/or in vivo application of the present invention. In certainpreferred embodiments, particle dimension (e.g., diameter)characterization is based on measurements using dynamic laser scattering(DLS). Mention is made of U.S. Pat. Nos. 8,709,843; 6,007,845;5,855,913; 5,985,309; 5,543,158; and the publication by James E. Dahlmanand Carmen Barnes et al. Nature Nanotechnology (2014) published online11 May 2014, doi:10.1038/nnano.2014.84, concerning particles, methods ofmaking and using them and measurements thereof. See also Dahlman et al.“Orthogonal gene control with a catalytically active Cas9 nuclease,”Nature Biotechnology 33, 1159-1161 (November, 2015)

Particles delivery systems within the scope of the present invention maybe provided in any form, including but not limited to solid, semi-solid,emulsion, or colloidal particles. As such any of the delivery systemsdescribed herein, including but not limited to, e.g., lipid-basedsystems, liposomes, micelles, microvesicles, exosomes, or gene gun maybe provided as particle delivery systems within the scope of the presentinvention.

Particles

Cas13b mRNA and guide RNA or crRNA may be delivered simultaneously usingparticles or lipid envelopes; for instance, CRISPR enzyme and RNA of theinvention, e.g., as a complex, can be delivered via a particle as inDahlman et al., WO2015089419 A2 and documents cited therein, such as 7C1(see, e.g., James E. Dahlman and Carmen Barnes et al. NatureNanotechnology (2014) published online 11 May 2014,doi:10.1038/nnano.2014.84), e.g., delivery particle comprising lipid orlipidoid and hydrophilic polymer, e.g., cationic lipid and hydrophilicpolymer, for instance wherein the cationic lipid comprises1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC) and/or whereinthe hydrophilic polymer comprises ethylene glycol or polyethylene glycol(PEG); and/or wherein the particle further comprises cholesterol (e.g.,particle from formulation 1=DOTAP 100, DMPC 0, PEG 0, Cholesterol 0;formulation number 2=DOTAP 90, DMPC 0, PEG 10, Cholesterol 0;formulation number 3=DOTAP 90, DMPC 0, PEG 5, Cholesterol 5), whereinparticles are formed using an efficient, multistep process whereinfirst, effector protein and RNA are mixed together, e.g., at a 1:1 molarratio, e.g., at room temperature, e.g., for 30 minutes, e.g., insterile, nuclease free 1×PBS; and separately, DOTAP, DMPC, PEG, andcholesterol as applicable for the formulation are dissolved in alcohol,e.g., 100% ethanol; and, the two solutions are mixed together to formparticles containing the complexes). Cas13b effector protein mRNA andguide RNA may be delivered simultaneously using particles or lipidenvelopes. This Dahlman et al technology can be applied in the instantinvention. An epoxide-modified lipid-polymer may be utilized to deliverthe nucleic acid-targeting system of the present invention to pulmonary,cardiovascular or renal cells, however, one of skill in the art mayadapt the system to deliver to other target organs. Dosage ranging fromabout 0.05 to about 0.6 mg/kg are envisioned. Dosages over several daysor weeks are also envisioned, with a total dosage of about 2 mg/kg. Forexample, Su X, Fricke J, Kavanagh D G, Irvine D J (“In vitro and in vivomRNA delivery using lipid-enveloped pH-responsive polymer nanoparticles”Mol Pharm. 2011 Jun. 6; 8(3):774-87. doi: 10.1021/mp100390w. Epub 2011Apr. 1) describes biodegradable core-shell structured particles with apoly(β-amino ester) (PBAE) core enveloped by a phospholipid bilayershell. These were developed for in vivo mRNA delivery. The pH-responsivePBAE component was chosen to promote endosome disruption, while thelipid surface layer was selected to minimize toxicity of the polycationcore. Such are, therefore, preferred for delivering RNA of the presentinvention.

In one embodiment, particles based on self-assembling bioadhesivepolymers are contemplated, which may be applied to oral delivery ofpeptides, intravenous delivery of peptides and nasal delivery ofpeptides, all to the brain. Other embodiments, such as oral absorptionand ocular delivery of hydrophobic drugs are also contemplated. Themolecular envelope technology involves an engineered polymer envelopewhich is protected and delivered to the site of the disease (see, e.g.,Mazza, M. et al. ACSNano, 2013. 7(2): 1016-1026; Siew, A., et al. MolPharm, 2012. 9(1):14-28; Lalatsa, A., et al. J Contr Rel, 2012.161(2):523-36; Lalatsa, A., et al., Mol Pharm, 2012. 9(6):1665-80;Lalatsa, A., et al. Mol Pharm, 2012. 9(6):1764-74; Garrett, N. L., etal. J Biophotonics, 2012. 5(5-6):458-68; Garrett, N. L., et al. J RamanSpect, 2012. 43(5):681-688; Ahmad, S., et al. J Royal Soc Interface2010. 7:S423-33; Uchegbu, I. F. Expert Opin Drug Deliv, 2006.3(5):629-40; Qu, X., et al. Biomacromolecules, 2006. 7(12):3452-9 andUchegbu, I. F., et al. Int J Pharm, 2001. 224:185-199). Doses of about 5mg/kg are contemplated, with single or multiple doses, depending on thetarget tissue.

Regarding particles, see, also Alabi et al., Proc Natl Acad Sci USA.2013 Aug. 6; 110(32):12881-6; Zhang et al., Adv Mater. 2013 Sep. 6;25(33):4641-5; Jiang et al., Nano Lett. 2013 Mar. 13; 13(3):1059-64;Karagiannis et al., ACS Nano. 2012 Oct. 23; 6(10):8484-7; Whitehead etal., ACS Nano. 2012 Aug. 28; 6(8):6922-9 and Lee et al., NatNanotechnol. 2012 Jun. 3; 7(6):389-93.

US patent application 20110293703 relates to lipidoid compounds are alsoparticularly useful in the administration of polynucleotides, which maybe applied to deliver the nucleic acid-targeting system of the presentinvention. In one aspect, the aminoalcohol lipidoid compounds arecombined with an agent to be delivered to a cell or a subject to formmicroparticles, nanoparticles, liposomes, or micelles. The agent to bedelivered by the particles, liposomes, or micelles may be in the form ofa gas, liquid, or solid, and the agent may be a polynucleotide, protein,peptide, or small molecule. The minoalcohol lipidoid compounds may becombined with other aminoalcohol lipidoid compounds, polymers (syntheticor natural), surfactants, cholesterol, carbohydrates, proteins, lipids,etc. to form the particles. These particles may then optionally becombined with a pharmaceutical excipient to form a pharmaceuticalcomposition. US Patent Publication No. 20110293703 also provides methodsof preparing the aminoalcohol lipidoid compounds. One or moreequivalents of an amine are allowed to react with one or moreequivalents of an epoxide-terminated compound under suitable conditionsto form an aminoalcohol lipidoid compound of the present invention. Incertain embodiments, all the amino groups of the amine are fully reactedwith the epoxide-terminated compound to form tertiary amines. In otherembodiments, all the amino groups of the amine are not fully reactedwith the epoxide-terminated compound to form tertiary amines therebyresulting in primary or secondary amines in the aminoalcohol lipidoidcompound. These primary or secondary amines are left as is or may bereacted with another electrophile such as a different epoxide-terminatedcompound. As will be appreciated by one skilled in the art, reacting anamine with less than excess of epoxide-terminated compound will resultin a plurality of different aminoalcohol lipidoid compounds with variousnumbers of tails. Certain amines may be fully functionalized with twoepoxide-derived compound tails while other molecules will not becompletely functionalized with epoxide-derived compound tails. Forexample, a diamine or polyamine may include one, two, three, or fourepoxide-derived compound tails off the various amino moieties of themolecule resulting in primary, secondary, and tertiary amines. Incertain embodiments, all the amino groups are not fully functionalized.In certain embodiments, two of the same types of epoxide-terminatedcompounds are used. In other embodiments, two or more differentepoxide-terminated compounds are used. The synthesis of the aminoalcohollipidoid compounds is performed with or without solvent, and thesynthesis may be performed at higher temperatures ranging from 30−100°C., preferably at approximately 50-90° C. The prepared aminoalcohollipidoid compounds may be optionally purified. For example, the mixtureof aminoalcohol lipidoid compounds may be purified to yield anaminoalcohol lipidoid compound with a particular number ofepoxide-derived compound tails. Or the mixture may be purified to yielda particular stereo- or regioisomer. The aminoalcohol lipidoid compoundsmay also be alkylated using an alkyl halide (e.g., methyl iodide) orother alkylating agent, and/or they may be acylated.

US Patent Publication No. 20110293703 also provides libraries ofaminoalcohol lipidoid compounds prepared by the inventive methods. Theseaminoalcohol lipidoid compounds may be prepared and/or screened usinghigh-throughput techniques involving liquid handlers, robots, microtiterplates, computers, etc. In certain embodiments, the aminoalcohollipidoid compounds are screened for their ability to transfectpolynucleotides or other agents (e.g., proteins, peptides, smallmolecules) into the cell. US Patent Publication No. 20130302401 relatesto a class of poly(beta-amino alcohols) (PBAAs) has been prepared usingcombinatorial polymerization. The inventive PBAAs may be used inbiotechnology and biomedical applications as coatings (such as coatingsof films or multilayer films for medical devices or implants),additives, materials, excipients, non-biofouling agents, micropatterningagents, and cellular encapsulation agents. When used as surfacecoatings, these PBAAs elicited different levels of inflammation, both invitro and in vivo, depending on their chemical structures. The largechemical diversity of this class of materials allowed us to identifypolymer coatings that inhibit macrophage activation in vitro.Furthermore, these coatings reduce the recruitment of inflammatorycells, and reduce fibrosis, following the subcutaneous implantation ofcarboxylated polystyrene microparticles. These polymers may be used toform polyelectrolyte complex capsules for cell encapsulation. Theinvention may also have many other biological applications such asantimicrobial coatings, DNA or siRNA delivery, and stem cell tissueengineering. The teachings of US Patent Publication No. 20130302401 maybe applied to the nucleic acid-targeting system of the presentinvention.

In another embodiment, lipid nanoparticles (LNPs) are contemplated. Anantitransthyretin small interfering RNA has been encapsulated in lipidnanoparticles and delivered to humans (see, e.g., Coelho et al., N EnglJ Med 2013; 369:819-29), and such a system may be adapted and applied tothe nucleic acid-targeting system of the present invention. Doses ofabout 0.01 to about 1 mg per kg of body weight administeredintravenously are contemplated. Medications to reduce the risk ofinfusion-related reactions are contemplated, such as dexamethasone,acetampinophen, diphenhydramine or cetirizine, and ranitidine arecontemplated. Multiple doses of about 0.3 mg per kilogram every 4 weeksfor five doses are also contemplated. LNPs have been shown to be highlyeffective in delivering siRNAs to the liver (see, e.g., Tabernero etal., Cancer Discovery, April 2013, Vol. 3, No. 4, pages 363-470) and aretherefore contemplated for delivering RNA encoding nucleicacid-targeting effector protein to the liver. A dosage of about fourdoses of 6 mg/kg of the LNP every two weeks may be contemplated.Tabernero et al. demonstrated that tumor regression was observed afterthe first 2 cycles of LNPs dosed at 0.7 mg/kg, and by the end of 6cycles the patient had achieved a partial response with completeregression of the lymph node metastasis and substantial shrinkage of theliver tumors. A complete response was obtained after 40 doses in thispatient, who has remained in remission and completed treatment afterreceiving doses over 26 months. Two patients with RCC and extrahepaticsites of disease including kidney, lung, and lymph nodes that wereprogressing following prior therapy with VEGF pathway inhibitors hadstable disease at all sites for approximately 8 to 12 months, and apatient with PNET and liver metastases continued on the extension studyfor 18 months (36 doses) with stable disease. However, the charge of theLNP must be taken into consideration. As cationic lipids combined withnegatively charged lipids to induce nonbilayer structures thatfacilitate intracellular delivery. Because charged LNPs are rapidlycleared from circulation following intravenous injection, ionizablecationic lipids with pKa values below 7 were developed (see, e.g., Rosinet al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, December2011). Negatively charged polymers such as RNA may be loaded into LNPsat low pH values (e.g., pH 4) where the ionizable lipids display apositive charge. However, at physiological pH values, the LNPs exhibit alow surface charge compatible with longer circulation times. Fourspecies of ionizable cationic lipids have been focused upon, namely1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinKDMA), and1,2-dilinoleyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane (DLinKC2-DMA). Ithas been shown that LNP siRNA systems containing these lipids exhibitremarkably different gene silencing properties in hepatocytes in vivo,with potencies varying according to the seriesDLinKC2-DMA>DLinKDMA>DLinDMA>>DLinDAP employing a Factor VII genesilencing model (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no.12, pages 1286-2200, December 2011). A dosage of 1 μg/ml of LNP orCRISPR-Cas RNA in or associated with the LNP may be contemplated,especially for a formulation containing DLinKC2-DMA.

Preparation of LNPs and CRISPR-Cas13b encapsulation may be used/and oradapted from Rosin et al, Molecular Therapy, vol. 19, no. 12, pages1286-2200, December 2011). The cationic lipids1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA),1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA),(3-o-[2″-(methoxypolyethyleneglycol 2000)succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG), andR-3-[(w-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-C-DOMG) may be providedby Tekmira Pharmaceuticals (Vancouver, Canada) or synthesized.Cholesterol may be purchased from Sigma (St Louis, Mo.). The specificnucleic acid-targeting complex (CRISPR-Cas) RNA may be encapsulated inLNPs containing DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationiclipid:DSPC:CHOL:PEGS-DMG or PEG-C-DOMG at 40:10:40:10 molar ratios).When required, 0.2% SP-DiOC18 (Invitrogen, Burlington, Canada) may beincorporated to assess cellular uptake, intracellular delivery, andbiodistribution. Encapsulation may be performed by dissolving lipidmixtures comprised of cationic lipid:DSPC:cholesterol:PEG-c-DOMG(40:10:40:10 molar ratio) in ethanol to a final lipid concentration of10 mmol/1. This ethanol solution of lipid may be added drop-wise to 50mmol/1 citrate, pH 4.0 to form multilamellar vesicles to produce a finalconcentration of 30% ethanol vol/vol. Large unilamellar vesicles may beformed following extrusion of multilamellar vesicles through two stacked80 nm Nuclepore polycarbonate filters using the Extruder (NorthernLipids, Vancouver, Canada). Encapsulation may be achieved by adding RNAdissolved at 2 mg/ml in 50 mmol/1 citrate, pH 4.0 containing 30% ethanolvol/vol drop-wise to extruded preformed large unilamellar vesicles andincubation at 31° C. for 30 minutes with constant mixing to a finalRNA/lipid weight ratio of 0.06/1 wt/wt. Removal of ethanol andneutralization of formulation buffer were performed by dialysis againstphosphate-buffered saline (PBS), pH 7.4 for 16 hours using Spectra/Por 2regenerated cellulose dialysis membranes. Particle size distribution maybe determined by dynamic light scattering using a NICOMP 370 particlesizer, the vesicle/intensity modes, and Gaussian fitting (NicompParticle Sizing, Santa Barbara, Calif.). The particle size for all threeLNP systems may be ˜70 nm in diameter. RNA encapsulation efficiency maybe determined by removal of free RNA using VivaPureD MiniH columns(Sartorius Stedim Biotech) from samples collected before and afterdialysis. The encapsulated RNA may be extracted from the elutedparticles and quantified at 260 nm. RNA to lipid ratio was determined bymeasurement of cholesterol content in vesicles using the Cholesterol Eenzymatic assay from Wako Chemicals USA (Richmond, Va.). In conjunctionwith the herein discussion of LNPs and PEG lipids, PEGylated liposomesor LNPs are likewise suitable for delivery of a nucleic acid-targetingsystem or components thereof. Preparation of large LNPs may be used/andor adapted from Rosin et al, Molecular Therapy, vol. 19, no. 12, pages1286-2200, December 2011. A lipid premix solution (20.4 mg/ml totallipid concentration) may be prepared in ethanol containing DLinKC2-DMA,DSPC, and cholesterol at 50:10:38.5 molar ratios. Sodium acetate may beadded to the lipid premix at a molar ratio of 0.75:1 (sodiumacetate:DLinKC2-DMA). The lipids may be subsequently hydrated bycombining the mixture with 1.85 volumes of citrate buffer (10 mmol/1, pH3.0) with vigorous stirring, resulting in spontaneous liposome formationin aqueous buffer containing 35% ethanol. The liposome solution may beincubated at 37° C. to allow for time-dependent increase in particlesize. Aliquots may be removed at various times during incubation toinvestigate changes in liposome size by dynamic light scattering(Zetasizer Nano Z S, Malvern Instruments, Worcestershire, UK). Once thedesired particle size is achieved, an aqueous PEG lipid solution(stock=10 mg/ml PEG-DMG in 35% (vol/vol) ethanol) may be added to theliposome mixture to yield a final PEG molar concentration of 3.5% oftotal lipid. Upon addition of PEG-lipids, the liposomes should theirsize, effectively quenching further growth. RNA may then be added to theempty liposomes at a RNA to total lipid ratio of approximately 1:10(wt:wt), followed by incubation for 30 minutes at 37° C. to form loadedLNPs. The mixture may be subsequently dialyzed overnight in PBS andfiltered with a 0.45-μm syringe filter.

Spherical Nucleic Acid (SNA™) constructs and other particles(particularly gold particles) are also contemplated as a means todelivery nucleic acid-targeting system to intended targets. Significantdata show that AuraSense Therapeutics' Spherical Nucleic Acid (SNA™)constructs, based upon nucleic acid-functionalized gold particles, areuseful.

Literature that may be employed in conjunction with herein teachingsinclude: Cutler et al., J. Am. Chem. Soc. 2011 133:9254-9257, Hao etal., Small. 2011 7:3158-3162, Zhang et al., ACS Nano. 2011 5:6962-6970,Cutler et al., J. Am. Chem. Soc. 2012 134:1376-1391, Young et al., NanoLett. 2012 12:3867-71, Zheng et al., Proc. Natl. Acad. Sci. USA. 2012109:11975-80, Mirkin, Nanomedicine 2012 7:635-638 Zhang et al., J. Am.Chem. Soc. 2012 134:16488-1691, Weintraub, Nature 2013 495:S14-S16, Choiet al., Proc. Natl. Acad. Sci. USA. 2013 110(19):7625-7630, Jensen etal., Sci. Transl. Med. 5, 209ra152 (2013) and Mirkin, et al., Small,10:186-192.

Self-assembling particles with RNA may be constructed withpolyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD)peptide ligand attached at the distal end of the polyethylene glycol(PEG). This system has been used, for example, as a means to targettumor neovasculature expressing integrins and deliver siRNA inhibitingvascular endothelial growth factor receptor-2 (VEGF R2) expression andthereby achieve tumor angiogenesis (see, e.g., Schiffelers et al.,Nucleic Acids Research, 2004, Vol. 32, No. 19). Nanoplexes may beprepared by mixing equal volumes of aqueous solutions of cationicpolymer and nucleic acid to give a net molar excess of ionizablenitrogen (polymer) to phosphate (nucleic acid) over the range of 2 to 6.The electrostatic interactions between cationic polymers and nucleicacid resulted in the formation of polyplexes with average particle sizedistribution of about 100 nm, hence referred to here as nanoplexes. Adosage of about 100 to 200 mg of nucleic acid-targeting complex RNA isenvisioned for delivery in the self-assembling particles of Schiffelerset al.

The nanoplexes of Bartlett et al. (PNAS, Sep. 25, 2007, vol. 104, no.39) may also be applied to the present invention. The nanoplexes ofBartlett et al. are prepared by mixing equal volumes of aqueoussolutions of cationic polymer and nucleic acid to give a net molarexcess of ionizable nitrogen (polymer) to phosphate (nucleic acid) overthe range of 2 to 6. The electrostatic interactions between cationicpolymers and nucleic acid resulted in the formation of polyplexes withaverage particle size distribution of about 100 nm, hence referred tohere as nanoplexes. The DOTA-siRNA of Bartlett et al. was synthesized asfollows: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acidmono(N-hydroxysuccinimide ester) (DOTA-NHSester) was ordered fromMacrocyclics (Dallas, Tex.). The amine modified RNA sense strand with a100-fold molar excess of DOTA-NHS-ester in carbonate buffer (pH 9) wasadded to a microcentrifuge tube. The contents were reacted by stirringfor 4 h at room temperature. The DOTA-RNAsense conjugate wasethanol-precipitated, resuspended in water, and annealed to theunmodified antisense strand to yield DOTA-siRNA. All liquids werepretreated with Chelex-100 (Bio-Rad, Hercules, Calif.) to remove tracemetal contaminants. Tf-targeted and nontargeted siRNA particles may beformed by using cyclodextrin-containing polycations. Typically,particles were formed in water at a charge ratio of 3 (+/−) and an siRNAconcentration of 0.5 g/liter. One percent of the adamantane-PEGmolecules on the surface of the targeted particles were modified with Tf(adamantane-PEG-Tf). The particles were suspended in a 5% (wt/vol)glucose carrier solution for injection.

Davis et al. (Nature, Vol 464, 15 Apr. 2010) conducts a RNA clinicaltrial that uses a targeted particle-delivery system (clinical trialregistration number NCT00689065). Patients with solid cancers refractoryto standard-of-care therapies are administered doses of targetedparticles on days 1, 3, 8 and 10 of a 21-day cycle by a 30-minintravenous infusion. The particles comprise, consist essentially of, orconsist of a synthetic delivery system containing: (1) a linear,cyclodextrin-based polymer (CDP), (2) a human transferrin protein (TF)targeting ligand displayed on the exterior of the nanoparticle to engageTF receptors (TFR) on the surface of the cancer cells, (3) a hydrophilicpolymer (polyethylene glycol (PEG) used to promote nanoparticlestability in biological fluids), and (4) siRNA designed to reduce theexpression of the RRM2 (sequence used in the clinic was previouslydenoted siR2B+5). The TFR has long been known to be upregulated inmalignant cells, and RRM2 is an established anti-cancer target. Theseparticles (clinical version denoted as CALAA-01) have been shown to bewell tolerated in multi-dosing studies in non-human primates. Although asingle patient with chronic myeloid leukaemia has been administeredsiRNAby liposomal delivery, Davis et al.'s clinical trial is the initialhuman trial to systemically deliver siRNA with a targeted deliverysystem and to treat patients with solid cancer. To ascertain whether thetargeted delivery system can provide effective delivery of functionalsiRNA to human tumours, Davis et al. investigated biopsies from threepatients from three different dosing cohorts; patients A, B and C, allof whom had metastatic melanoma and received CALAA-01 doses of 18, 24and 30 mg m⁻² siRNA, respectively. Similar doses may also becontemplated for the nucleic acid-targeting system of the presentinvention. The delivery of the invention may be achieved with particlescontaining a linear, cyclodextrin-based polymer (CDP), a humantransferrin protein (TF) targeting ligand displayed on the exterior ofthe particle to engage TF receptors (TFR) on the surface of the cancercells and/or a hydrophilic polymer (for example, polyethylene glycol(PEG) used to promote particle stability in biological fluids).

In terms of this invention, it is preferred to have one or morecomponents of RNA-targeting complex, e.g., nucleic acid-targetingeffector (Cas13b) protein or mRNA therefor, or guide RNA or crRNAdelivered using particles or lipid envelopes. Other delivery systems orvectors are may be used in conjunction with the particle aspects of theinvention. Particles encompassed in the present invention may beprovided in different forms, e.g., as solid particles (e.g., metal suchas silver, gold, iron, titanium), non-metal, lipid-based solids,polymers), suspensions of particles, or combinations thereof. Metal,dielectric, and semiconductor particles may be prepared, as well ashybrid structures (e.g., core-shell particles). Particles made ofsemiconducting material may also be labeled quantum dots if they aresmall enough (typically sub 10 nm) that quantization of electronicenergy levels occurs. Such nanoscale particles are used in biomedicalapplications as drug carriers or imaging agents and may be adapted forsimilar purposes in the present invention.

Semi-solid and soft particles have been manufactured, and are within thescope of the present invention. A prototype particle of semi-solidnature is the liposome. Various types of liposome particles arecurrently used clinically as delivery systems for anticancer drugs andvaccines. Particles with one half hydrophilic and the other halfhydrophobic are termed Janus particles and are particularly effectivefor stabilizing emulsions. They can self-assemble at water/oilinterfaces and act as solid surfactants.

U.S. Pat. No. 8,709,843, incorporated herein by reference, provides adrug delivery system for targeted delivery of therapeuticagent-containing particles to tissues, cells, and intracellularcompartments. The invention provides targeted particles comprisingpolymer conjugated to a surfactant, hydrophilic polymer or lipid. U.S.Pat. No. 6,007,845, incorporated herein by reference, provides particleswhich have a core of a multiblock copolymer formed by covalently linkinga multifunctional compound with one or more hydrophobic polymers and oneor more hydrophilic polymers, and contain a biologically activematerial. U.S. Pat. No. 5,855,913, incorporated herein by reference,provides a particulate composition having aerodynamically lightparticles having a tap density of less than 0.4 g/cm3 with a meandiameter of between 5 μm and 30 μm, incorporating a surfactant on thesurface thereof for drug delivery to the pulmonary system. U.S. Pat. No.5,985,309, incorporated herein by reference, provides particlesincorporating a surfactant and/or a hydrophilic or hydrophobic complexof a positively or negatively charged therapeutic or diagnostic agentand a charged molecule of opposite charge for delivery to the pulmonarysystem. U.S. Pat. No. 5,543,158, incorporated herein by reference,provides biodegradable injectable particles having a biodegradable solidcore containing a biologically active material and poly(alkylene glycol)moieties on the surface. WO2012135025 (also published as US20120251560),incorporated herein by reference, describes conjugated polyethyleneimine(PEI) polymers and conjugated aza-macrocycles (collectively referred toas “conjugated lipomer” or “lipomers”). In certain embodiments, it canbe envisioned that such methods and materials of herein-cited documents,e.g., conjugated lipomers can be used in the context of the nucleicacid-targeting system to achieve in vitro, ex vivo and in vivo genomicperturbations to modify gene expression, including modulation of proteinexpression.

Exosomes

Exosomes are endogenous nano-vesicles that transport RNAs and proteins,and which can deliver RNA to the brain and other target organs. Toreduce immunogenicity, Alvarez-Erviti et al. (2011, Nat Biotechnol 29:341) used self-derived dendritic cells for exosome production. Targetingto the brain was achieved by engineering the dendritic cells to expressLamp2b, an exosomal membrane protein, fused to the neuron-specific RVGpeptide. Purified exosomes were loaded with exogenous RNA byelectroporation. Intravenously injected RVG-targeted exosomes deliveredGAPDH siRNA specifically to neurons, microglia, oligodendrocytes in thebrain, resulting in a specific gene knockdown. Pre-exposure to RVGexosomes did not attenuate knockdown, and non-specific uptake in othertissues was not observed. The therapeutic potential of exosome-mediatedsiRNA delivery was demonstrated by the strong mRNA (60%) and protein(62%) knockdown of BACE1, a therapeutic target in Alzheimer's disease.

To obtain a pool of immunologically inert exosomes, Alvarez-Erviti etal. harvested bone marrow from inbred C57BL/6 mice with a homogenousmajor histocompatibility complex (MHC) haplotype. As immature dendriticcells produce large quantities of exosomes devoid of T-cell activatorssuch as MHC-II and CD86, Alvarez-Erviti et al. selected for dendriticcells with granulocyte/macrophage-colony stimulating factor (GM-CSF) for7 d. Exosomes were purified from the culture supernatant the followingday using well-established ultracentrifugation protocols. The exosomesproduced were physically homogenous, with a size distribution peaking at80 nm in diameter as determined by particle tracking analysis (NTA) andelectron microscopy. Alvarez-Erviti et al. obtained 6-12 μg of exosomes(measured based on protein concentration) per 10⁶ cells. Next,Alvarez-Erviti et al. investigated the possibility of loading modifiedexosomes with exogenous cargoes using electroporation protocols adaptedfor nanoscale applications. As electroporation for membrane particles atthe nanometer scale is not well-characterized, nonspecific Cy5-labeledRNA was used for the empirical optimization of the electroporationprotocol. The amount of encapsulated RNA was assayed afterultracentrifugation and lysis of exosomes. Electroporation at 400 V and125 μF resulted in the greatest retention of RNA and was used for allsubsequent experiments. Alvarez-Erviti et al. administered 150 μg ofeach BACE1 siRNA encapsulated in 150 μg of RVG exosomes to normalC57BL/6 mice and compared the knockdown efficiency to four controls:untreated mice, mice injected with RVG exosomes only, mice injected withBACE1 siRNA complexed to an in vivo cationic liposome reagent and miceinjected with BACE1 siRNA complexed to RVG-9R, the RVG peptideconjugated to 9 D-arginines that electrostatically binds to the siRNA.Cortical tissue samples were analyzed 3 d after administration and asignificant protein knockdown (45%, P<0.05, versus 62%, P<0.01) in bothsiRNA-RVG-9R-treated and siRNARVG exosome-treated mice was observed,resulting from a significant decrease in BACE1 mRNA levels (66% [+ or −]15%, P<0.001 and 61% [+ or −] 13% respectively, P<0.01). Moreover,Applicants demonstrated a significant decrease (55%, P<0.05) in thetotal [beta]-amyloid 1-42 levels, a main component of the amyloidplaques in Alzheimer's pathology, in the RVG-exosome-treated animals.The decrease observed was greater than the β-amyloid 1-40 decreasedemonstrated in normal mice after intraventricular injection of BACE1inhibitors. Alvarez-Erviti et al. carried out 5′-rapid amplification ofcDNA ends (RACE) on BACE1 cleavage product, which provided evidence ofRNAi-mediated knockdown by the siRNA. Finally, Alvarez-Erviti et al.investigated whether RNA-RVG exosomes induced immune responses in vivoby assessing IL-6, IP-10, TNFα and IFN-α serum concentrations. Followingexosome treatment, nonsignificant changes in all cytokines wereregistered similar to siRNA-transfection reagent treatment in contrastto siRNA-RVG-9R, which potently stimulated IL-6 secretion, confirmingthe immunologically inert profile of the exosome treatment. Given thatexosomes encapsulate only 20% of siRNA, delivery with RVG-exosomeappears to be more efficient than RVG-9R delivery as comparable mRNAknockdown and greater protein knockdown was achieved with fivefold lesssiRNA without the corresponding level of immune stimulation. Thisexperiment demonstrated the therapeutic potential of RVG-exosometechnology, which is potentially suited for long-term silencing of genesrelated to neurodegenerative diseases. The exosome delivery system ofAlvarez-Erviti et al. may be applied to deliver the nucleicacid-targeting system of the present invention to therapeutic targets,especially neurodegenerative diseases. A dosage of about 100 to 1000 mgof nucleic acid-targeting system encapsulated in about 100 to 1000 mg ofRVG exosomes may be contemplated for the present invention.

El-Andaloussi et al. (Nature Protocols 7, 2112-2126(2012)) providesexosomes derived from cultured cells harnessed for delivery of RNA invitro and in vivo. This protocol first describes the generation oftargeted exosomes through transfection of an expression vector,comprising an exosomal protein fused with a peptide ligand. Next,El-Andaloussi et al. explain how to purify and characterize exosomesfrom transfected cell supernatant. Next, El-Andaloussi et al. detailcrucial steps for loading RNA into exosomes. Finally, El-Andaloussi etal. outline how to use exosomes to efficiently deliver RNA in vitro andin vivo in mouse brain. Examples of anticipated results in whichexosome-mediated RNA delivery is evaluated by functional assays andimaging are also provided. The entire protocol takes ˜3 weeks. Deliveryor administration according to the invention may be performed usingexosomes produced from self-derived dendritic cells. From the hereinteachings, this can be employed in the practice of the invention

In another embodiment, the plasma exosomes of Wahlgren et al. (NucleicAcids Research, 2012, Vol. 40, No. 17 e130) are contemplated. Exosomesare nano-sized vesicles (30-90 nm in size) produced by many cell types,including dendritic cells (DC), B cells, T cells, mast cells, epithelialcells and tumor cells. These vesicles are formed by inward budding oflate endosomes and are then released to the extracellular environmentupon fusion with the plasma membrane. Because exosomes naturally carryRNA between cells, this property may be useful in gene therapy, and fromthis disclosure can be employed in the practice of the instantinvention. Exosomes from plasma can be prepared by centrifugation ofbuffy coat at 900 g for 20 min to isolate the plasma followed byharvesting cell supernatants, centrifuging at 300 g for 10 min toeliminate cells and at 16 500 g for 30 min followed by filtrationthrough a 0.22 mm filter. Exosomes are pelleted by ultracentrifugationat 120 000 g for 70 min. Chemical transfection of siRNA into exosomes iscarried out according to the manufacturer's instructions in RNAiHuman/Mouse Starter Kit (Quiagen, Hilden, Germany). siRNA is added to100 ml PBS at a final concentration of 2 mmol/ml. After adding HiPerFecttransfection reagent, the mixture is incubated for 10 min at RT. Inorder to remove the excess of micelles, the exosomes are re-isolatedusing aldehyde/sulfate latex beads. The chemical transfection of nucleicacid-targeting system into exosomes may be conducted similarly to siRNA.The exosomes may be co-cultured with monocytes and lymphocytes isolatedfrom the peripheral blood of healthy donors. Therefore, it may becontemplated that exosomes containing nucleic acid-targeting system maybe introduced to monocytes and lymphocytes of and autologouslyreintroduced into a human. Accordingly, delivery or administrationaccording to the invention may be performed using plasma exosomes.

Liposomes

Delivery or administration according to the invention can be performedwith liposomes. Liposomes are spherical vesicle structures composed of auni- or multilamellar lipid bilayer surrounding internal aqueouscompartments and a relatively impermeable outer lipophilic phospholipidbilayer. Liposomes have gained considerable attention as drug deliverycarriers because they are biocompatible, nontoxic, can deliver bothhydrophilic and lipophilic drug molecules, protect their cargo fromdegradation by plasma enzymes, and transport their load acrossbiological membranes and the blood brain barrier (BBB) (see, e.g., Spuchand Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12pages, 2011. doi:10.1155/2011/469679 for review). Liposomes can be madefrom several different types of lipids; however, phospholipids are mostcommonly used to generate liposomes as drug carriers. Although liposomeformation is spontaneous when a lipid film is mixed with an aqueoussolution, it can also be expedited by applying force in the form ofshaking by using a homogenizer, sonicator, or an extrusion apparatus(see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011,Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679 for review).

Several other additives may be added to liposomes in order to modifytheir structure and properties. For instance, either cholesterol orsphingomyelin may be added to the liposomal mixture in order to helpstabilize the liposomal structure and to prevent the leakage of theliposomal inner cargo. Further, liposomes are prepared from hydrogenatedegg phosphatidylcholine or egg phosphatidylcholine, cholesterol, anddicetyl phosphate, and their mean vesicle sizes were adjusted to about50 and 100 nm. (see, e.g., Spuch and Navarro, Journal of Drug Delivery,vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679for review). A liposome formulation may be mainly comprised of naturalphospholipids and lipids such as1,2-distearoryl-sn-glycero-3-phosphatidyl choline (DSPC), sphingomyelin,egg phosphatidylcholines and monosialoganglioside. Since thisformulation is made up of phospholipids only, liposomal formulationshave encountered many challenges, one of the ones being the instabilityin plasma. Several attempts to overcome these challenges have been made,specifically in the manipulation of the lipid membrane. One of theseattempts focused on the manipulation of cholesterol. Addition ofcholesterol to conventional formulations reduces rapid release of theencapsulated bioactive compound into the plasma or1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) increases thestability (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol.2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679 forreview). In a particularly advantageous embodiment, Trojan Horseliposomes (also known as Molecular Trojan Horses) are desirable andprotocols may be found athttp://cshprotocols.cshop.org/content/2010/4/pdb.prot5407.long. Theseparticles allow delivery of a transgene to the entire brain after anintravascular injection. Without being bound by limitation, it isbelieved that neutral lipid particles with specific antibodiesconjugated to surface allow crossing of the blood brain barrier viaendocytosis. Applicant postulates utilizing Trojan Horse Liposomes todeliver the CRISPR-Cas13b complexes to the brain via an intravascularinjection, which would allow whole brain transgenic animals without theneed for embryonic manipulation. About 1-5 g of DNA or RNA may becontemplated for in vivo administration in liposomes.

In another embodiment, the nucleic acid-targeting system or componentsthereof may be administered in liposomes, such as a stablenucleic-acid-lipid particle (SNALP) (see, e.g., Morrissey et al., NatureBiotechnology, Vol. 23, No. 8, August 2005). Daily intravenousinjections of about 1, 3 or 5 mg/kg/day of a specific nucleicacid-targeting system targeted in a SNALP are contemplated. The dailytreatment may be over about three days and then weekly for about fiveweeks. In another embodiment, a specific nucleic acid-targeting systemencapsulated SNALP) administered by intravenous injection to at doses ofabout 1 or 2.5 mg/kg are also contemplated (see, e.g., Zimmerman et al.,Nature Letters, Vol. 441, 4 May 2006). The SNALP formulation may containthe lipids 3-N-[(wmethoxypoly(ethylene glycol) 2000)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA),1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, in a2:40:10:48 molar percent ratio (see, e.g., Zimmerman et al., NatureLetters, Vol. 441, 4 May 2006). In another embodiment, stablenucleic-acid-lipid particles (SNALPs) have proven to be effectivedelivery molecules to highly vascularized HepG2-derived liver tumors butnot in poorly vascularized HCT-116 derived liver tumors (see, e.g., Li,Gene Therapy (2012) 19, 775-780). The SNALP liposomes may be prepared byformulating D-Lin-DMA and PEG-C-DMA with distearoylphosphatidylcholine(DSPC), Cholesterol and siRNA using a 25:1 lipid/siRNA ratio and a48/40/10/2 molar ratio of Cholesterol/D-Lin-DMA/DSPC/PEG-C-DMA. Theresulted SNALP liposomes are about 80-100 nm in size. In yet anotherembodiment, a SNALP may comprise synthetic cholesterol (Sigma-Aldrich,St Louis, Mo., USA), dipalmitoylphosphatidylcholine (Avanti PolarLipids, Alabaster, Ala., USA), 3-N-[(w-methoxy poly(ethyleneglycol)2000)carbamoyl]-1,2-dimyrestyloxypropylamine, and cationic1,2-dilinoleyloxy-3-N,Ndimethylaminopropane (see, e.g., Geisbert et al.,Lancet 2010; 375: 1896-905). A dosage of about 2 mg/kg total nucleicacid-targeting systemper dose administered as, for example, a bolusintravenous infusion may be contemplated. In yet another embodiment, aSNALP may comprise synthetic cholesterol (Sigma-Aldrich),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti Polar LipidsInc.), PEG-cDMA, and 1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane(DLinDMA) (see, e.g., Judge, J. Clin. Invest. 119:661-673 (2009)).Formulations used for in vivo studies may comprise a final lipid/RNAmass ratio of about 9:1.

The safety profile of RNAi nanomedicines has been reviewed by Barros andGollob of Alnylam Pharmaceuticals (see, e.g., Advanced Drug DeliveryReviews 64 (2012) 1730-1737). The stable nucleic acid lipid particle(SNALP) is comprised of four different lipids—an ionizable lipid(DLinDMA) that is cationic at low pH, a neutral helper lipid,cholesterol, and a diffusible polyethylene glycol (PEG)-lipid. Theparticle is approximately 80 nm in diameter and is charge-neutral atphysiologic pH. During formulation, the ionizable lipid serves tocondense lipid with the anionic RNA during particle formation. Whenpositively charged under increasingly acidic endosomal conditions, theionizable lipid also mediates the fusion of SNALP with the endosomalmembrane enabling release of RNA into the cytoplasm. The PEG-lipidstabilizes the particle and reduces aggregation during formulation, andsubsequently provides a neutral hydrophilic exterior that improvespharmacokinetic properties. To date, two clinical programs have beeninitiated using SNALP formulations with RNA. Tekmira Pharmaceuticalsrecently completed a phase I single-dose study of SNALP-ApoB in adultvolunteers with elevated LDL cholesterol. ApoB is predominantlyexpressed in the liver and jejunum and is essential for the assembly andsecretion of VLDL and LDL. Seventeen subjects received a single dose ofSNALP-ApoB (dose escalation across 7 dose levels). There was no evidenceof liver toxicity (anticipated as the potential dose-limiting toxicitybased on preclinical studies). One (of two) subjects at the highest doseexperienced flu-like symptoms consistent with immune system stimulation,and the decision was made to conclude the trial. Alnylam Pharmaceuticalshas similarly advanced ALN-TTR01, which employs the SNALP technologydescribed above and targets hepatocyte production of both mutant andwild-type TTR to treat TTR amyloidosis (ATTR). Three ATTR syndromes havebeen described: familial amyloidotic polyneuropathy (FAP) and familialamyloidotic cardiomyopathy (FAC)—both caused by autosomal dominantmutations in TTR; and senile systemic amyloidosis (SSA) cause bywildtype TTR. A placebo-controlled, single dose-escalation phase I trialof ALN-TTR01 was recently completed in patients with ATTR. ALN-TTR01 wasadministered as a 15-minute IV infusion to 31 patients (23 with studydrug and 8 with placebo) within a dose range of 0.01 to 1.0 mg/kg (basedon siRNA). Treatment was well tolerated with no significant increases inliver function tests. Infusion-related reactions were noted in 3 of 23patients at ≥0.4 mg/kg; all responded to slowing of the infusion rateand all continued on study. Minimal and transient elevations of serumcytokines IL-6, IP-10 and IL-lra were noted in two patients at thehighest dose of 1 mg/kg (as anticipated from preclinical and NHPstudies). Lowering of serum TTR, the expected pharmacodynamics effect ofALN-TTR01, was observed at 1 mg/kg.

In yet another embodiment, a SNALP may be made by solubilizing acationic lipid, DSPC, cholesterol and PEG-lipid e.g., in ethanol, e.g.,at a molar ratio of 40:10:40:10, respectively (see, Semple et al.,Nature Niotechnology, Volume 28 Number 2 Feb. 2010, pp. 172-177). Thelipid mixture was added to an aqueous buffer (50 mM citrate, pH 4) withmixing to a final ethanol and lipid concentration of 30% (vol/vol) and6.1 mg/ml, respectively, and allowed to equilibrate at 22° C. for 2 minbefore extrusion. The hydrated lipids were extruded through two stacked80 nm pore-sized filters (Nuclepore) at 22° C. using a Lipex Extruder(Northern Lipids) until a vesicle diameter of 70-90 nm, as determined bydynamic light scattering analysis, was obtained. This generally required1-3 passes. The siRNA (solubilized in a 50 mM citrate, pH 4 aqueoussolution containing 30% ethanol) was added to the pre-equilibrated (35°C.) vesicles at a rate of ˜5 ml/min with mixing. After a final targetsiRNA/lipid ratio of 0.06 (wt/wt) was reached, the mixture was incubatedfor a further 30 min at 35° C. to allow vesicle reorganization andencapsulation of the siRNA. The ethanol was then removed and theexternal buffer replaced with PBS (155 mM NaCl, 3 mM Na₂HPO₄, 1 mMKH₂PO₄, pH 7.5) by either dialysis or tangential flow diafiltration.siRNA were encapsulated in SNALP using a controlled step-wise dilutionmethod process. The lipid constituents of KC2-SNALP were DLin-KC2-DMA(cationic lipid), dipalmitoylphosphatidylcholine (DPPC; Avanti PolarLipids), synthetic cholesterol (Sigma) and PEG-C-DMA used at a molarratio of 57.1:7.1:34.3:1.4. Upon formation of the loaded particles,SNALP were dialyzed against PBS and filter sterilized through a 0.2 μmfilter before use. Mean particle sizes were 75-85 nm and 90-95% of thesiRNA was encapsulated within the lipid particles. The final siRNA/lipidratio in formulations used for in vivo testing was ˜0.15 (wt/wt).LNP-siRNA systems containing Factor VII siRNA were diluted to theappropriate concentrations in sterile PBS immediately before use and theformulations were administered intravenously through the lateral tailvein in a total volume of 10 ml/kg. This method and these deliverysystems may be extrapolated to the nucleic acid-targeting system of thepresent invention.

Other Lipids

Other cationic lipids, such as amino lipid2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) maybe utilized to encapsulate nucleic acid-targeting system or componentsthereof or nucleic acid molecule(s) coding therefor e.g., similar toSiRNA (see, e.g., Jayaraman, Angew. Chem. Int. Ed. 2012, 51, 8529-8533),and hence may be employed in the practice of the invention. A preformedvesicle with the following lipid composition may be contemplated: aminolipid, distearoylphosphatidylcholine (DSPC), cholesterol and(R)-2,3-bis(octadecyloxy) propyl-1-(methoxy poly(ethyleneglycol)2000)propylcarbamate (PEG-lipid) in the molar ratio 40/10/40/10,respectively, and a FVII siRNA/total lipid ratio of approximately 0.05(w/w). To ensure a narrow particle size distribution in the range of70-90 nm and a low polydispersity index of 0.11+0.04 (n=56), theparticles may be extruded up to three times through 80 nm membranesprior to adding the guide RNA. Particles containing the highly potentamino lipid 16 may be used, in which the molar ratio of the four lipidcomponents 16, DSPC, cholesterol and PEG-lipid (50/10/38.5/1.5) whichmay be further optimized to enhance in vivo activity.

Michael S D Kormann et al. (“Expression of therapeutic proteins afterdelivery of chemically modified mRNA in mice: Nature Biotechnology,Volume:29, Pages: 154-157 (2011)) describes the use of lipid envelopesto deliver RNA. Use of lipid envelopes is also preferred in the presentinvention.

In another embodiment, lipids may be formulated with the RNA-targetingsystem (CRISPR-Cas13b complex, i.e., the Cas13b complexed with crRNA) ofthe present invention or component(s) thereof or nucleic acidmolecule(s) coding therefor to form lipid nanoparticles (LNPs). Lipidsinclude, but are not limited to, DLin-KC2-DMA4, C12-200 and colipidsdisteroylphosphatidyl choline, cholesterol, and PEG-DMG may beformulated with RNA-targeting system instead of siRNA (see, e.g.,Novobrantseva, Molecular Therapy-Nucleic Acids (2012) 1, e4;doi:10.1038/mtna.2011.3) using a spontaneous vesicle formationprocedure. The component molar ratio may be about 50/10/38.5/1.5(DLin-KC2-DMA or C12-200/disteroylphosphatidylcholine/cholesterol/PEG-DMG). The final lipid:siRNA weight ratio may be˜12:1 and 9:1 in the case of DLin-KC2-DMA and C12-200 lipid particles(LNPs), respectively. The formulations may have mean particle diametersof ˜80 nm with >90% entrapment efficiency. A 3 mg/kg dose may becontemplated. Tekmira has a portfolio of approximately 95 patentfamilies, in the U.S. and abroad, that are directed to various aspectsof LNPs and LNP formulations (see, e.g., U.S. Pat. Nos. 7,982,027;7,799,565; 8,058,069; 8,283,333; 7,901,708; 7,745,651; 7,803,397;8,101,741; 8,188,263; 7,915,399; 8,236,943 and 7,838,658 and EuropeanPat. Nos 1766035; 1519714; 1781593 and 1664316), all of which may beused and/or adapted to the present invention.

The RNA-targeting system or components thereof or nucleic acidmolecule(s) coding therefor may be delivered encapsulated in PLGAMicrospheres such as that further described in US published applications20130252281 and 20130245107 and 20130244279 (assigned to ModernaTherapeutics) which relate to aspects of formulation of compositionscomprising modified nucleic acid molecules which may encode a protein, aprotein precursor, or a partially or fully processed form of the proteinor a protein precursor. The formulation may have a molar ratio50:10:38.5:1.5-3.0 (cationic lipid:fusogenic lipid:cholesterol:PEGlipid). The PEG lipid may be selected from, but is not limited toPEG-c-DOMG, PEG-DMG. The fusogenic lipid may be DSPC. See also, Schrumet al., Delivery and Formulation of Engineered Nucleic Acids, USpublished application 20120251618.

Nanomerics' technology addresses bioavailability challenges for a broadrange of therapeutics, including low molecular weight hydrophobic drugs,peptides, and nucleic acid based therapeutics (plasmid, siRNA, miRNA).Specific administration routes for which the technology has demonstratedclear advantages include the oral route, transport across theblood-brain-barrier, delivery to solid tumours, as well as to the eye.See, e.g., Mazza et al., 2013, ACS Nano. 2013 Feb. 26; 7(2):1016-26;Uchegbu and Siew, 2013, J Pharm Sci. 102(2):305-10 and Lalatsa et al.,2012, J Control Release. 2012 Jul. 20; 161(2):523-36.

US Patent Publication No. 20050019923 describes cationic dendrimers fordelivering bioactive molecules, such as polynucleotide molecules,peptides and polypeptides and/or pharmaceutical agents, to a mammalianbody. The dendrimers are suitable for targeting the delivery of thebioactive molecules to, for example, the liver, spleen, lung, kidney orheart (or even the brain). Dendrimers are synthetic 3-dimensionalmacromolecules that are prepared in a step-wise fashion from simplebranched monomer units, the nature and functionality of which can beeasily controlled and varied. Dendrimers are synthesized from therepeated addition of building blocks to a multifunctional core(divergent approach to synthesis), or towards a multifunctional core(convergent approach to synthesis) and each addition of a 3-dimensionalshell of building blocks leads to the formation of a higher generationof the dendrimers. Polypropylenimine dendrimers start from adiaminobutane core to which is added twice the number of amino groups bya double Michael addition of acrylonitrile to the primary aminesfollowed by the hydrogenation of the nitriles. This results in adoubling of the amino groups. Polypropylenimine dendrimers contain 100%protonable nitrogens and up to 64 terminal amino groups (generation 5,DAB 64). Protonable groups are usually amine groups which are able toaccept protons at neutral pH. The use of dendrimers as gene deliveryagents has largely focused on the use of the polyamidoamine. andphosphorous containing compounds with a mixture of amine/amide orN—P(O₂)S as the conjugating units respectively with no work beingreported on the use of the lower generation polypropylenimine dendrimersfor gene delivery. Polypropylenimine dendrimers have also been studiedas pH sensitive controlled release systems for drug delivery and fortheir encapsulation of guest molecules when chemically modified byperipheral amino acid groups. The cytotoxicity and interaction ofpolypropylenimine dendrimers with DNA as well as the transfectionefficacy of DAB 64 has also been studied. US Patent Publication No.20050019923 is based upon the observation that, contrary to earlierreports, cationic dendrimers, such as polypropylenimine dendrimers,display suitable properties, such as specific targeting and lowtoxicity, for use in the targeted delivery of bioactive molecules, suchas genetic material. In addition, derivatives of the cationic dendrimeralso display suitable properties for the targeted delivery of bioactivemolecules. See also, Bioactive Polymers, US published application20080267903, which discloses “Various polymers, including cationicpolyamine polymers and dendrimeric polymers, are shown to possessanti-proliferative activity, and may therefore be useful for treatmentof disorders characterised by undesirable cellular proliferation such asneoplasms and tumours, inflammatory disorders (including autoimmunedisorders), psoriasis and atherosclerosis. The polymers may be usedalone as active agents, or as delivery vehicles for other therapeuticagents, such as drug molecules or nucleic acids for gene therapy. Insuch cases, the polymers' own intrinsic anti-tumour activity maycomplement the activity of the agent to be delivered.” The disclosuresof these patent publications may be employed in conjunction with hereinteachings for delivery of nucleic acid-targetingsystem(s) orcomponent(s) thereof or nucleic acid molecule(s) coding therefor.

Supercharged Proteins

Supercharged proteins are a class of engineered or naturally occurringproteins with unusually high positive or negative net theoretical chargeand may be employed in delivery of nucleic acid-targetingsystem(s) orcomponent(s) thereof or nucleic acid molecule(s) coding therefor. Bothsupernegatively and superpositively charged proteins exhibit aremarkable ability to withstand thermally or chemically inducedaggregation. Superpositively charged proteins are also able to penetratemammalian cells. Associating cargo with these proteins, such as plasmidDNA, RNA, or other proteins, can enable the functional delivery of thesemacromolecules into mammalian cells both in vitro and in vivo. DavidLiu's lab reported the creation and characterization of superchargedproteins in 2007 (Lawrence et al., 2007, Journal of the AmericanChemical Society 129, 10110-10112).

The nonviral delivery of RNA and plasmid DNA into mammalian cells arevaluable both for research and therapeutic applications (Akinc et al.,2010, Nat. Biotech. 26, 561-569). Purified +36 GFP protein (or othersuperpositively charged protein) is mixed with RNAs in the appropriateserum-free media and allowed to complex prior addition to cells.Inclusion of serum at this stage inhibits formation of the superchargedprotein-RNA complexes and reduces the effectiveness of the treatment.The following protocol has been found to be effective for a variety ofcell lines (McNaughton et al., 2009, Proc. Natl. Acad. Sci. USA 106,6111-6116). However, pilot experiments varying the dose of protein andRNA should be performed to optimize the procedure for specific celllines. (1) One day before treatment, plate 1×10⁵ cells per well in a48-well plate. (2) On the day of treatment, dilute purified +36 GFPprotein in serumfree media to a final concentration 200 nM. Add RNA to afinal concentration of 50 nM. Vortex to mix and incubate at roomtemperature for 10 min. (3) During incubation, aspirate media from cellsand wash once with PBS. (4) Following incubation of +36 GFP and RNA, addthe protein-RNA complexes to cells. (5) Incubate cells with complexes at37° C. for 4h. (6) Following incubation, aspirate the media and washthree times with 20 U/mL heparin PBS. Incubate cells withserum-containing media for a further 48h or longer depending upon theassay for activity. (7) Analyze cells by immunoblot, qPCR, phenotypicassay, or other appropriate method.

+36 GFP was found to be an effective plasmid delivery reagent in a rangeof cells. See also, e.g., McNaughton et al., Proc. Natl. Acad. Sci. USA106, 6111-6116 (2009); Cronican et al., ACS Chemical Biology 5, 747-752(2010); Cronican et al., Chemistry & Biology 18, 833-838 (2011);Thompson et al., Methods in Enzymology 503, 293-319 (2012); Thompson, D.B., et al., Chemistry & Biology 19 (7), 831-843 (2012). The methods ofthe super charged proteins may be used and/or adapted for delivery ofthe RNA-targeting system(s) or component(s) thereof or nucleic acidmolecule(s) coding therefor of the invention.

Cell Penetrating Peptides (CPPs)

In yet another embodiment, cell penetrating peptides (CPPs) arecontemplated for the delivery of the CRISPR Cas system. CPPs are shortpeptides that facilitate cellular uptake of various molecular cargo(from nanosize particles to small chemical molecules and large fragmentsof DNA). The term “cargo” as used herein includes but is not limited tothe group consisting of therapeutic agents, diagnostic probes, peptides,nucleic acids, antisense oligonucleotides, plasmids, proteins, particlesincluding nanoparticles, liposomes, chromophores, small molecules andradioactive materials. In aspects of the invention, the cargo may alsocomprise any component of the CRISPR Cas system or the entire functionalCRISPR Cas system. Aspects of the present invention further providemethods for delivering a desired cargo into a subject comprising: (a)preparing a complex comprising the cell penetrating peptide of thepresent invention and a desired cargo, and (b) orally, intraarticularly,intraperitoneally, intrathecally, intrarterially, intranasally,intraparenchymally, subcutaneously, intramuscularly, intravenously,dermally, intrarectally, or topically administering the complex to asubject. The cargo is associated with the peptides either throughchemical linkage via covalent bonds or through non-covalentinteractions. The function of the CPPs are to deliver the cargo intocells, a process that commonly occurs through endocytosis with the cargodelivered to the endosomes of living mammalian cells. Cell-penetratingpeptides are of different sizes, amino acid sequences, and charges butall CPPs have one distinct characteristic, which is the ability totranslocate the plasma membrane and facilitate the delivery of variousmolecular cargoes to the cytoplasm or an organelle. CPP translocationmay be classified into three main entry mechanisms: direct penetrationin the membrane, endocytosis-mediated entry, and translocation throughthe formation of a transitory structure. CPPs have found numerousapplications in medicine as drug delivery agents in the treatment ofdifferent diseases including cancer and virus inhibitors, as well ascontrast agents for cell labeling. Examples of the latter include actingas a carrier for GFP, MM contrast agents, or quantum dots. CPPs holdgreat potential as in vitro and in vivo delivery vectors for use inresearch and medicine. CPPs typically have an amino acid compositionthat either contains a high relative abundance of positively chargedamino acids such as lysine or arginine or has sequences that contain analternating pattern of polar/charged amino acids and non-polar,hydrophobic amino acids. These two types of structures are referred toas polycationic or amphipathic, respectively. A third class of CPPs arethe hydrophobic peptides, containing only apolar residues, with low netcharge or have hydrophobic amino acid groups that are crucial forcellular uptake. One of the initial CPPs discovered was thetrans-activating transcriptional activator (Tat) from HumanImmunodeficiency Virus 1 (HIV-1) which was found to be efficiently takenup from the surrounding media by numerous cell types in culture. Sincethen, the number of known CPPs has expanded considerably and smallmolecule synthetic analogues with more effective protein transductionproperties have been generated. CPPs include but are not limited toPenetratin, Tat (48-60), Transportan, and (R-AhX-R4)(Ahx=aminohexanoyl).

U.S. Pat. No. 8,372,951, provides a CPP derived from eosinophil cationicprotein (ECP) which exhibits highly cell-penetrating efficiency and lowtoxicity. Aspects of delivering the CPP with its cargo into a vertebratesubject are also provided. Further aspects of CPPs and their deliveryare described in U.S. Pat. Nos. 8,575,305; 8; 614,194 and 8,044,019.CPPs can be used to deliver the CRISPR-Cas system or components thereof.That CPPs can be employed to deliver the CRISPR-Cas system or componentsthereof is also provided in the manuscript “Gene disruption bycell-penetrating peptide-mediated delivery of Cas9 protein and guideRNA”, by Suresh Ramakrishna, Abu-Bonsrah Kwaku Dad, Jagadish Beloor, etal. Genome Res. 2014 Apr. 2. [Epub ahead of print], incorporated byreference in its entirety, wherein it is demonstrated that treatmentwith CPP-conjugated recombinant Cas9 protein and CPP-complexed guideRNAs lead to endogenous gene disruptions in human cell lines. In thepaper the Cas9 protein was conjugated to CPP via a thioether bond,whereas the guide RNA was complexed with CPP, forming condensed,positively charged particles. It was shown that simultaneous andsequential treatment of human cells, including embryonic stem cells,dermal fibroblasts, HEK293T cells, HeLa cells, and embryonic carcinomacells, with the modified Cas9 and guide RNA led to efficient genedisruptions with reduced off-target mutations relative to plasmidtransfections. CPP delivery can be used in the practice of theinvention.

Implantable Devices

In another embodiment, implantable devices are also contemplated fordelivery of the nucleic acid-targeting system or component(s) thereof ornucleic acid molecule(s) coding therefor. For example, US PatentPublication 20110195123 discloses an implantable medical device whichelutes a drug locally and in prolonged period is provided, includingseveral types of such a device, the treatment modes of implementationand methods of implantation. The device comprising of polymericsubstrate, such as a matrix for example, that is used as the devicebody, and drugs, and in some cases additional scaffolding materials,such as metals or additional polymers, and materials to enhancevisibility and imaging. An implantable delivery device can beadvantageous in providing release locally and over a prolonged period,where drug is released directly to the extracellular matrix (ECM) of thediseased area such as tumor, inflammation, degeneration or forsymptomatic objectives, or to injured smooth muscle cells, or forprevention. One kind of drug is RNA, as disclosed above, and this systemmay be used/and or adapted to the nucleic acid-targeting system of thepresent invention. The modes of implantation in some embodiments areexisting implantation procedures that are developed and used today forother treatments, including brachytherapy and needle biopsy. In suchcases the dimensions of the new implant described in this invention aresimilar to the original implant. Typically a few devices are implantedduring the same treatment procedure.

US Patent Publication 20110195123, provides a drug delivery implantableor insertable system, including systems applicable to a cavity such asthe abdominal cavity and/or any other type of administration in whichthe drug delivery system is not anchored or attached, comprising abiostable and/or degradable and/or bioabsorbable polymeric substrate,which may for example optionally be a matrix. It should be noted thatthe term “insertion” also includes implantation. The drug deliverysystem is preferably implemented as a “Loder” as described in US PatentPublication 20110195123.

The polymer or plurality of polymers are biocompatible, incorporating anagent and/or plurality of agents, enabling the release of agent at acontrolled rate, wherein the total volume of the polymeric substrate,such as a matrix for example, in some embodiments is optionally andpreferably no greater than a maximum volume that permits a therapeuticlevel of the agent to be reached. As a non-limiting example, such avolume is preferably within the range of 0.1 m³ to 1000 mm³, as requiredby the volume for the agent load. The Loder may optionally be larger,for example when incorporated with a device whose size is determined byfunctionality, for example and without limitation, a knee joint, anintra-uterine or cervical ring and the like.

The drug delivery system (for delivering the composition) is designed insome embodiments to preferably employ degradable polymers, wherein themain release mechanism is bulk erosion; or in some embodiments, nondegradable, or slowly degraded polymers are used, wherein the mainrelease mechanism is diffusion rather than bulk erosion, so that theouter part functions as membrane, and its internal part functions as adrug reservoir, which practically is not affected by the surroundingsfor an extended period (for example from about a week to about a fewmonths). Combinations of different polymers with different releasemechanisms may also optionally be used. The concentration gradient atthe surface is preferably maintained effectively constant during asignificant period of the total drug releasing period, and therefore thediffusion rate is effectively constant (termed “zero mode” diffusion).By the term “constant” it is meant a diffusion rate that is preferablymaintained above the lower threshold of therapeutic effectiveness, butwhich may still optionally feature an initial burst and/or mayfluctuate, for example increasing and decreasing to a certain degree.The diffusion rate is preferably so maintained for a prolonged period,and it can be considered constant to a certain level to optimize thetherapeutically effective period, for example the effective silencingperiod.

The drug delivery system optionally and preferably is designed to shieldthe nucleotide based therapeutic agent from degradation, whetherchemical in nature or due to attack from enzymes and other factors inthe body of the subject.

The drug delivery system of US Patent Publication 20110195123 isoptionally associated with sensing and/or activation appliances that areoperated at and/or after implantation of the device, by non and/orminimally invasive methods of activation and/oracceleration/deceleration, for example optionally including but notlimited to thermal heating and cooling, laser beams, and ultrasonic,including focused ultrasound and/or RF (radiofrequency) methods ordevices.

According to some embodiments of US Patent Publication 20110195123, thesite for local delivery may optionally include target sitescharacterized by high abnormal proliferation of cells, and suppressedapoptosis, including tumors, active and or chronic inflammation andinfection including autoimmune diseases states, degenerating tissueincluding muscle and nervous tissue, chronic pain, degenerative sites,and location of bone fractures and other wound locations for enhancementof regeneration of tissue, and injured cardiac, smooth and striatedmuscle.

The site for implantation of the composition, or target site, preferablyfeatures a radius, area and/or volume that is sufficiently small fortargeted local delivery. For example, the target site optionally has adiameter in a range of from about 0.1 mm to about 5 cm.

The location of the target site is preferably selected for maximumtherapeutic efficacy. For example, the composition of the drug deliverysystem (optionally with a device for implantation as described above) isoptionally and preferably implanted within or in the proximity of atumor environment, or the blood supply associated thereof.

For example the composition (optionally with the device) is optionallyimplanted within or in the proximity to pancreas, prostate, breast,liver, via the nipple, within the vascular system and so forth.

The target location is optionally selected from the group comprising,consisting essentially of, or consisting of (as non-limiting examplesonly, as optionally any site within the body may be suitable forimplanting a Loder): 1. brain at degenerative sites like in Parkinson orAlzheimer disease at the basal ganglia, white and gray matter; 2. spineas in the case of amyotrophic lateral sclerosis (ALS); 3. uterine cervixto prevent HPV infection; 4. active and chronic inflammatory joints; 5.dermis as in the case of psoriasis; 6. sympathetic and sensoric nervoussites for analgesic effect; 7. Intra osseous implantation; 8. acute andchronic infection sites; 9. Intra vaginal; 10. Inner ear—auditorysystem, labyrinth of the inner ear, vestibular system; 11. Intratracheal; 12. Intra-cardiac; coronary, epicardiac; 13. urinary bladder;14. biliary system; 15. parenchymal tissue including and not limited tothe kidney, liver, spleen; 16. lymph nodes; 17. salivary glands; 18.dental gums; 19. Intra-articular (into joints); 20. Intra-ocular; 21.Brain tissue; 22. Brain ventricles; 23. Cavities, including abdominalcavity (for example but without limitation, for ovary cancer); 24. Intraesophageal and 25. Intra rectal.

Optionally insertion of the system (for example a device containing thecomposition) is associated with injection of material to the ECM at thetarget site and the vicinity of that site to affect local pH and/ortemperature and/or other biological factors affecting the diffusion ofthe drug and/or drug kinetics in the ECM, of the target site and thevicinity of such a site.

Optionally, according to some embodiments, the release of said agentcould be associated with sensing and/or activation appliances that areoperated prior and/or at and/or after insertion, by non and/or minimallyinvasive and/or else methods of activation and/oracceleration/deceleration, including laser beam, radiation, thermalheating and cooling, and ultrasonic, including focused ultrasound and/orRF (radiofrequency) methods or devices, and chemical activators.

According to other embodiments of US Patent Publication 20110195123, thedrug preferably comprises a RNA, for example for localized cancer casesin breast, pancreas, brain, kidney, bladder, lung, and prostate asdescribed below. Although exemplified with RNAi, many drugs areapplicable to be encapsulated in Loder, and can be used in associationwith this invention, as long as such drugs can be encapsulated with theLoder substrate, such as a matrix for example, and this system may beused and/or adapted to deliver the nucleic acid-targeting system of thepresent invention.

As another example of a specific application, neuro and musculardegenerative diseases develop due to abnormal gene expression. Localdelivery of RNAs may have therapeutic properties for interfering withsuch abnormal gene expression. Local delivery of anti apoptotic, antiinflammatory and anti degenerative drugs including small drugs andmacromolecules may also optionally be therapeutic. In such cases theLoder is applied for prolonged release at constant rate and/or through adedicated device that is implanted separately. All of this may be usedand/or adapted to the nucleic acid-targeting system of the presentinvention.

As yet another example of a specific application, psychiatric andcognitive disorders are treated with gene modifiers. Gene knockdown is atreatment option. Loders locally delivering agents to central nervoussystem sites are therapeutic options for psychiatric and cognitivedisorders including but not limited to psychosis, bi-polar diseases,neurotic disorders and behavioral maladies. The Loders could alsodeliver locally drugs including small drugs and macromolecules uponimplantation at specific brain sites. All of this may be used and/oradapted to the nucleic acid-targeting system of the present invention.

As another example of a specific application, silencing of innate and/oradaptive immune mediators at local sites enables the prevention of organtransplant rejection. Local delivery of RNAs and immunomodulatingreagents with the Loder implanted into the transplanted organ and/or theimplanted site renders local immune suppression by repelling immunecells such as CD8 activated against the transplanted organ. All of thismay be used/and or adapted to the nucleic acid-targeting system of thepresent invention.

As another example of a specific application, vascular growth factorsincluding VEGFs and angiogenin and others are essential forneovascularization. Local delivery of the factors, peptides,peptidomimetics, or suppressing their repressors is an importanttherapeutic modality; silencing the repressors and local delivery of thefactors, peptides, macromolecules and small drugs stimulatingangiogenesis with the Loder is therapeutic for peripheral, systemic andcardiac vascular disease.

The method of insertion, such as implantation, may optionally already beused for other types of tissue implantation and/or for insertions and/orfor sampling tissues, optionally without modifications, or alternativelyoptionally only with non-major modifications in such methods. Suchmethods optionally include but are not limited to brachytherapy methods,biopsy, endoscopy with and/or without ultrasound, such as ERCP,stereotactic methods into the brain tissue, Laparoscopy, includingimplantation with a laparoscope into joints, abdominal organs, thebladder wall and body cavities.

Implantable device technology herein discussed can be employed withherein teachings and hence by this disclosure and the knowledge in theart, CRISPR-Cas system or components thereof or nucleic acid moleculesthereof or encoding or providing components may be delivered via animplantable device.

Patient-Specific Screening Methods

A nucleic acid-targeting system that targets RNA, e.g., trinucleotiderepeats can be used to screen patients or patent samples for thepresence of such repeats. The repeats can be the target of the RNA ofthe nucleic acid-targeting system, and if there is binding thereto bythe nucleic acid-targeting system, that binding can be detected, tothereby indicate that such a repeat is present. Thus, a nucleicacid-targeting system can be used to screen patients or patient samplesfor the presence of the repeat. The patient can then be administeredsuitable compound(s) to address the condition; or, can be administered anucleic acid-targeting system to bind to and cause insertion, deletionor mutation and alleviate the condition.

The invention uses nucleic acids to bind target RNA sequences.

CRISPR Effector Protein mRNA and Guide RNA

CRISPR effector (Cas13b) protein or mRNA therefor (or more generally anucleuic acid molecule therefor) and guide RNA or crRNA might also bedelivered separately e.g., the former 1-12 hours (preferably around 2-6hours) prior to the administration of guide RNA or crRNA, or together. Asecond booster dose of guide RNA or crRNA can be administered 1-12 hours(preferably around 2-6 hours) after the initial administration.

The Cas13b effector protein is sometimes referred to herein as a CRISPREnzyme. It will be appreciated that the effector protein is based on orderived from an enzyme, so the term ‘effector protein’ certainlyincludes ‘enzyme’ in some embodiments. However, it will also beappreciated that the effector protein may, as required in someembodiments, have DNA or RNA binding, but not necessarily cutting ornicking, activity, including a dead-Cas effector protein function.

Cellular targets include Hemopoietic Stem/Progenitor Cells (CD34+);Human T cells; and Eye (retinal cells)—for example photoreceptorprecursor cells.

Inventive methods can further comprise delivery of templates. Deliveryof templates may be via the cotemporaneous or separate from delivery ofany or all the CRISPR effector protein (Cas13b) or guide or crRNA andvia the same delivery mechanism or different. Inducible Systems

In some embodiments, a CRISPR effector (Cas 13b) protein may form acomponent of an inducible system. The inducible nature of the systemwould allow for spatiotemporal control of gene editing or geneexpression using a form of energy. The form of energy may include but isnot limited to electromagnetic radiation, sound energy, chemical energyand thermal energy. Examples of inducible system include tetracyclineinducible promoters (Tet-On or Tet-Off), small molecule two-hybridtranscription activations systems (FKBP, ABA, etc), or light induciblesystems (Phytochrome, LOV domains, or cryptochrome). In one embodiment,the CRISPR effector protein may be a part of a Light InducibleTranscriptional Effector (LITE) to direct changes in transcriptionalactivity in a sequence-specific manner. The components of a light mayinclude a CRISPR effector protein, a light-responsive cytochromeheterodimer (e.g. from Arabidopsis thaliana), and a transcriptionalactivation/repression domain. Further examples of inducible DNA bindingproteins and methods for their use are provided in U.S. 61/736,465 andU.S. 61/721,283, and WO 2014018423 A2 which is hereby incorporated byreference in its entirety.

Self-Inactivating Systems

Once all copies of RNA in a cell have been edited, continued Cas13beffector protein expression or activity in that cell is no longernecessary. A Self-Inactivating system that relies on the use of RNA asto the Cas13b or crRNA as the guide target sequence can shut down thesystem by preventing expression of Cas13b or complex formation.

Kits

In one aspect, the invention provides kits containing any one or more ofthe elements disclosed in the above methods and compositions. In someembodiments, the kit comprises a vector system as taught herein or oneor more of the components of the CRISPR/Cas13b system or complex astaught herein, such as crRNAs and/or Cas13b effector protein or Cas13beffector protein encoding mRNA, and instructions for using the kit.Elements may be provide individually or in combinations, and may beprovided in any suitable container, such as a vial, a bottle, or a tube.In some embodiments, the kit includes instructions in one or morelanguages, for example in more than one language. The instructions maybe specific to the applications and methods described herein. In someembodiments, a kit comprises one or more reagents for use in a processutilizing one or more of the elements described herein. Reagents may beprovided in any suitable container. For example, a kit may provide oneor more reaction or storage buffers. Reagents may be provided in a formthat is usable in a particular assay, or in a form that requiresaddition of one or more other components before use (e.g., inconcentrate or lyophilized form). A buffer can be any buffer, includingbut not limited to a sodium carbonate buffer, a sodium bicarbonatebuffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer,and combinations thereof. In some embodiments, the buffer is alkaline.In some embodiments, the buffer has a pH from about 7 to about 10. Insome embodiments, the kit comprises one or more oligonucleotidescorresponding to a guide sequence for insertion into a vector so as tooperably link the guide or crRNA sequence and a regulatory element. Insome embodiments, the kit comprises a homologous recombination templatepolynucleotide. In some embodiments, the kit comprises one or more ofthe vectors and/or one or more of the polynucleotides described herein.The kit may advantageously allows to provide all elements of the systemsof the invention.

The invention has a broad spectrum of applications in, e.g., genetherapy, drug screening, disease diagnosis, and prognosis.

The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”,“nucleic acid” and “oligonucleotide” are used interchangeably. Theyrefer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three dimensional structure, and mayperform any function, known or unknown. The following are non-limitingexamples of polynucleotides: coding or non-coding regions of a gene orgene fragment, loci (locus) defined from linkage analysis, exons,introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, shortinterfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA),ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides,plasmids, vectors, isolated DNA of any sequence, isolated RNA of anysequence, nucleic acid probes, and primers. The term also encompassesnucleic-acid-like structures with synthetic backbones, see, e.g.,Eckstein, 1991; Baserga et al., 1992; Milligan, 1993; WO 97/03211; WO96/39154; Mata, 1997; Strauss-Soukup, 1997; and Samstag, 1996. Apolynucleotide may comprise one or more modified nucleotides, such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure may be imparted before or after assembly ofthe polymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.As used herein the term “wild type” is a term of the art understood byskilled persons and means the typical form of an organism, strain, geneor characteristic as it occurs in nature as distinguished from mutant orvariant forms. A “wild type” can be a base line. As used herein the term“variant” should be taken to mean the exhibition of qualities that havea pattern that deviates from what occurs in nature. The terms“non-naturally occurring” or “engineered” are used interchangeably andindicate the involvement of the hand of man. The terms, when referringto nucleic acid molecules or polypeptides mean that the nucleic acidmolecule or the polypeptide is at least substantially free from at leastone other component with which they are naturally associated in natureand as found in nature. “Complementarity” refers to the ability of anucleic acid to form hydrogen bond(s) with another nucleic acid sequenceby either traditional Watson-Crick base pairing or other non-traditionaltypes. A percent complementarity indicates the percentage of residues ina nucleic acid molecule which can form hydrogen bonds (e.g.,Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5,6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100%complementary). “Perfectly complementary” means that all the contiguousresidues of a nucleic acid sequence will hydrogen bond with the samenumber of contiguous residues in a second nucleic acid sequence.“Substantially complementary” as used herein refers to a degree ofcomplementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or morenucleotides, or refers to two nucleic acids that hybridize understringent conditions. As used herein, “stringent conditions” forhybridization refer to conditions under which a nucleic acid havingcomplementarity to a target sequence predominantly hybridizes with thetarget sequence, and substantially does not hybridize to non-targetsequences. Stringent conditions are generally sequence-dependent, andvary depending on a number of factors. In general, the longer thesequence, the higher the temperature at which the sequence specificallyhybridizes to its target sequence. Non-limiting examples of stringentconditions are described in detail in Tijssen (1993), LaboratoryTechniques In Biochemistry And Molecular Biology-Hybridization WithNucleic Acid Probes Part I, Second Chapter “Overview of principles ofhybridization and the strategy of nucleic acid probe assay”, Elsevier,N.Y. Where reference is made to a polynucleotide sequence, thencomplementary or partially complementary sequences are also envisaged.These are preferably capable of hybridizing to the reference sequenceunder highly stringent conditions. Generally, in order to maximize thehybridization rate, relatively low-stringency hybridization conditionsare selected: about 20 to 25° C. lower than the thermal melting point(T_(m)). The T_(m) is the temperature at which 50% of specific targetsequence hybridizes to a perfectly complementary probe in solution at adefined ionic strength and pH. Generally, in order to require at leastabout 85% nucleotide complementarity of hybridized sequences, highlystringent washing conditions are selected to be about 5 to 15° C. lowerthan the T_(m) In order to require at least about 70% nucleotidecomplementarity of hybridized sequences, moderately-stringent washingconditions are selected to be about 15 to 30° C. lower than the T_(m)Highly permissive (very low stringency) washing conditions may be as lowas 50° C. below the T_(m), allowing a high level of mis-matching betweenhybridized sequences. Those skilled in the art will recognize that otherphysical and chemical parameters in the hybridization and wash stagescan also be altered to affect the outcome of a detectable hybridizationsignal from a specific level of homology between target and probesequences. Preferred highly stringent conditions comprise incubation in50% formamide, 5×SSC, and 1% SDS at 42° C., or incubation in 5×SSC and1% SDS at 65° C., with wash in 0.2×SSC and 0.1% SDS at 65° C.“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson Crick base pairing, Hoogstein binding, or inany other sequence specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combinationof these. A hybridization reaction may constitute a step in a moreextensive process, such as the initiation of PCR, or the cleavage of apolynucleotide by an enzyme. A sequence capable of hybridizing with agiven sequence is referred to as the “complement” of the given sequence.As used herein, the term “genomic locus” or “locus” (plural loci) is thespecific location of a gene or DNA sequence on a chromosome. A “gene”refers to stretches of DNA or RNA that encode a polypeptide or an RNAchain that has functional role to play in an organism and hence is themolecular unit of heredity in living organisms. For the purpose of thisinvention it may be considered that genes include regions which regulatethe production of the gene product, whether or not such regulatorysequences are adjacent to coding and/or transcribed sequences.Accordingly, a gene includes, but is not necessarily limited to,promoter sequences, terminators, translational regulatory sequences suchas ribosome binding sites and internal ribosome entry sites, enhancers,silencers, insulators, boundary elements, replication origins, matrixattachment sites and locus control regions. As used herein, “expressionof a genomic locus” or “gene expression” is the process by whichinformation from a gene is used in the synthesis of a functional geneproduct. The products of gene expression are often proteins, but innon-protein coding genes such as rRNA genes or tRNA genes, the productis functional RNA. The process of gene expression is used by all knownlife—eukaryotes (including multicellular organisms), prokaryotes(bacteria and archaea) and viruses to generate functional products tosurvive. As used herein “expression” of a gene or nucleic acidencompasses not only cellular gene expression, but also thetranscription and translation of nucleic acid(s) in cloning systems andin any other context. As used herein, “expression” also refers to theprocess by which a polynucleotide is transcribed from a DNA template(such as into and mRNA or other RNA transcript) and/or the process bywhich a transcribed mRNA is subsequently translated into peptides,polypeptides, or proteins. Transcripts and encoded polypeptides may becollectively referred to as “gene product.” If the polynucleotide isderived from genomic DNA, expression may include splicing of the mRNA ina eukaryotic cell. The terms “polypeptide”, “peptide” and “protein” areused interchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation, such asconjugation with a labeling component. As used herein the term “aminoacid” includes natural and/or unnatural or synthetic amino acids,including glycine and both the D or L optical isomers, and amino acidanalogs and peptidomimetics. As used herein, the term “domain” or“protein domain” refers to a part of a protein sequence that may existand function independently of the rest of the protein chain. Asdescribed in aspects of the invention, sequence identity is related tosequence homology. Homology comparisons may be conducted by eye, or moreusually, with the aid of readily available sequence comparison programs.These commercially available computer programs may calculate percent (%)homology between two or more sequences and may also calculate thesequence identity shared by two or more amino acid or nucleic acidsequences.

As used herein the term “wild type” is a term of the art understood byskilled persons and means the typical form of an organism, strain, geneor characteristic as it occurs in nature as distinguished from mutant orvariant forms. A “wild type” can be a base line.

As used herein the term “variant” should be taken to mean the exhibitionof qualities that have a pattern that deviates from what occurs innature. The terms “non-naturally occurring” or “engineered” are usedinterchangeably and indicate the involvement of the hand of man. Theterms, when referring to nucleic acid molecules or polypeptides meanthat the nucleic acid molecule or the polypeptide is at leastsubstantially free from at least one other component with which they arenaturally associated in nature and as found in nature. In all aspectsand embodiments, whether they include these terms or not, it will beunderstood that, preferably, the may be optional and thus preferablyincluded or not preferably not included. Furthermore, the terms“non-naturally occurring” and “engineered” may be used interchangeablyand so can therefore be used alone or in combination and one or othermay replace mention of both together. In particular, “engineered” ispreferred in place of “non-naturally occurring” or “non-naturallyoccurring and/or engineered.”

Sequence homologies may be generated by any of a number of computerprograms known in the art, for example BLAST or FASTA, etc. A suitablecomputer program for carrying out such an alignment is the GCG WisconsinBestfit package (University of Wisconsin, U.S.A; Devereux et al., 1984,Nucleic Acids Research 12:387). Examples of other software than mayperform sequence comparisons include, but are not limited to, the BLASTpackage (see Ausubel et al., 1999 ibid—Chapter 18), FASTA (Atschul etal., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparisontools. Both BLAST and FASTA are available for offline and onlinesearching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). Howeverit is preferred to use the GCG Bestfit program. Percentage (%) sequencehomology may be calculated over contiguous sequences, i.e., one sequenceis aligned with the other sequence and each amino acid or nucleotide inone sequence is directly compared with the corresponding amino acid ornucleotide in the other sequence, one residue at a time. This is calledan “ungapped” alignment. Typically, such ungapped alignments areperformed only over a relatively short number of residues. Although thisis a very simple and consistent method, it fails to take intoconsideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion may cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without unduly penalizing the overall homology or identityscore. This is achieved by inserting “gaps” in the sequence alignment totry to maximize local homology or identity. However, these more complexmethods assign “gap penalties” to each gap that occurs in the alignmentso that, for the same number of identical amino acids, a sequencealignment with as few gaps as possible—reflecting higher relatednessbetween the two compared sequences—may achieve a higher score than onewith many gaps. “Affinity gap costs” are typically used that charge arelatively high cost for the existence of a gap and a smaller penaltyfor each subsequent residue in the gap. This is the most commonly usedgap scoring system. High gap penalties may, of course, produce optimizedalignments with fewer gaps. Most alignment programs allow the gappenalties to be modified. However, it is preferred to use the defaultvalues when using such software for sequence comparisons. For example,when using the GCG Wisconsin Bestfit package the default gap penalty foramino acid sequences is −12 for a gap and −4 for each extension.Calculation of maximum % homology therefore first requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (Devereux et al., 1984Nuc. Acids Research 12 p387). Examples of other software than mayperform sequence comparisons include, but are not limited to, the BLASTpackage (see Ausubel et al., 1999 Short Protocols in Molecular Biology,4^(th) Ed. —Chapter 18), FASTA (Altschul et al., 1990 J. Mol. Biol.403-410) and the GENEWORKS suite of comparison tools. Both BLAST andFASTA are available for offline and online searching (see Ausubel etal., 1999, Short Protocols in Molecular Biology, pages 7-58 to 7-60).However, for some applications, it is preferred to use the GCG Bestfitprogram. A new tool, called BLAST 2 Sequences is also available forcomparing protein and nucleotide sequences (see FEMS Microbiol Lett.1999 174(2): 247-50; FEMS Microbiol Lett. 1999 177(1): 187-8 and thewebsite of the National Center for Biotechnology information at thewebsite of the National Institutes for Health). Although the final %homology may be measured in terms of identity, the alignment processitself is typically not based on an all-or-nothing pair comparison.Instead, a scaled similarity score matrix is generally used that assignsscores to each pair-wise comparison based on chemical similarity orevolutionary distance. An example of such a matrix commonly used is theBLOSUM62 matrix—the default matrix for the BLAST suite of programs. GCGWisconsin programs generally use either the public default values or acustom symbol comparison table, if supplied (see user manual for furtherdetails). For some applications, it is preferred to use the publicdefault values for the GCG package, or in the case of other software,the default matrix, such as BLOSUM62. Alternatively, percentagehomologies may be calculated using the multiple alignment feature inDNASIS™ (Hitachi Software), based on an algorithm, analogous to CLUSTAL(Higgins D G & Sharp P M (1988), Gene 73(1), 237-244). Once the softwarehas produced an optimal alignment, it is possible to calculate %homology, preferably % sequence identity. The software typically doesthis as part of the sequence comparison and generates a numericalresult. The sequences may also have deletions, insertions orsubstitutions of amino acid residues which produce a silent change andresult in a functionally equivalent substance. Deliberate amino acidsubstitutions may be made on the basis of similarity in amino acidproperties (such as polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues) and it istherefore useful to group amino acids together in functional groups.Amino acids may be grouped together based on the properties of theirside chains alone. However, it is more useful to include mutation dataas well. The sets of amino acids thus derived are likely to be conservedfor structural reasons. These sets may be described in the form of aVenn diagram (Livingstone C. D. and Barton G. J. (1993) “Proteinsequence alignments: a strategy for the hierarchical analysis of residueconservation” Comput. Appl. Biosci. 9: 745-756) (Taylor W. R. (1986)“The classification of amino acid conservation” J. Theor. Biol. 119;205-218). Conservative substitutions may be made, for example accordingto the table below which describes a generally accepted Venn diagramgrouping of amino acids.

Set Sub-set Hydro- F W Y H K M I L V A Aromatic F W Y H phobicG C(SEQ ID NO: 132) Aliphatic I L V Polar W Y H K R E D C S T ChargedH K R E D N Q Positively H K R (SEQ ID NO: 133) charged Negatively E Dcharged Small V C A G S P T N D Tiny A G S (SEQ ID NO: 134)

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a vertebrate, preferably a mammal,more preferably a human. Mammals include, but are not limited to,murines, simians, humans, farm animals, sport animals, and pets.Tissues, cells and their progeny of a biological entity obtained in vivoor cultured in vitro are also encompassed.

The terms “therapeutic agent”, “therapeutic capable agent” or “treatmentagent” are used interchangeably and refer to a molecule or compound thatconfers some beneficial effect upon administration to a subject. Thebeneficial effect includes enablement of diagnostic determinations;amelioration of a disease, symptom, disorder, or pathological condition;reducing or preventing the onset of a disease, symptom, disorder orcondition; and generally counteracting a disease, symptom, disorder orpathological condition. As used herein, “treatment” or “treating,” or“palliating” or “ameliorating” are used interchangeably. These termsrefer to an approach for obtaining beneficial or desired resultsincluding but not limited to a therapeutic benefit and/or a prophylacticbenefit. By therapeutic benefit is meant any therapeutically relevantimprovement in or effect on one or more diseases, conditions, orsymptoms under treatment. For prophylactic benefit, the compositions maybe administered to a subject at risk of developing a particular disease,condition, or symptom, or to a subject reporting one or more of thephysiological symptoms of a disease, even though the disease, condition,or symptom may not have yet been manifested. The term “effective amount”or “therapeutically effective amount” refers to the amount of an agentthat is sufficient to effect beneficial or desired results. Thetherapeutically effective amount may vary depending upon one or more of:the subject and disease condition being treated, the weight and age ofthe subject, the severity of the disease condition, the manner ofadministration and the like, which can readily be determined by one ofordinary skill in the art. The term also applies to a dose that willprovide an image for detection by any one of the imaging methodsdescribed herein. The specific dose may vary depending on one or moreof: the particular agent chosen, the dosing regimen to be followed,whether it is administered in combination with other compounds, timingof administration, the tissue to be imaged, and the physical deliverysystem in which it is carried.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of immunology, biochemistry,chemistry, molecular biology, microbiology, cell biology, genomics andrecombinant DNA, which are within the skill of the art. See Sambrook,Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2ndedition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel,et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press,Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, ALABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).Several aspects of the invention relate to vector systems comprising oneor more vectors, or vectors as such. Vectors can be designed forexpression of CRISPR transcripts (e.g. nucleic acid transcripts,proteins, or enzymes) in prokaryotic or eukaryotic cells. For example,CRISPR transcripts can be expressed in bacterial cells such asEscherichia coli, insect cells (using baculovirus expression vectors),yeast cells, or mammalian cells. Suitable host cells are discussedfurther in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY185, Academic Press, San Diego, Calif. (1990). Alternatively, therecombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase. Embodiments of the invention include sequences (bothpolynucleotide or polypeptide) which may comprise homologoussubstitution (substitution and replacement are both used herein to meanthe interchange of an existing amino acid residue or nucleotide, with analternative residue or nucleotide) that may occur i.e., like-for-likesubstitution in the case of amino acids such as basic for basic, acidicfor acidic, polar for polar, etc. Non-homologous substitution may alsooccur i.e., from one class of residue to another or alternativelyinvolving the inclusion of unnatural amino acids such as ornithine(hereinafter referred to as Z), diaminobutyric acid ornithine(hereinafter referred to as B), norleucine ornithine (hereinafterreferred to as O), pyriylalanine, thienylalanine, naphthylalanine andphenylglycine. Variant amino acid sequences may include suitable spacergroups that may be inserted between any two amino acid residues of thesequence including alkyl groups such as methyl, ethyl or propyl groupsin addition to amino acid spacers such as glycine or β-alanine residues.A further form of variation, which involves the presence of one or moreamino acid residues in peptoid form, may be well understood by thoseskilled in the art. For the avoidance of doubt, “the peptoid form” isused to refer to variant amino acid residues wherein the α-carbonsubstituent group is on the residue's nitrogen atom rather than theα-carbon. Processes for preparing peptides in the peptoid form are knownin the art, for example Simon R J et al., PNAS (1992) 89(20), 9367-9371and Horwell D C, Trends Biotechnol. (1995) 13(4), 132-134. Homologymodelling: Corresponding residues in other Cas13b orthologs can beidentified by the methods of Zhang et al., 2012 (Nature; 490(7421):556-60) and Chen et al., 2015 (PLoS Comput Biol; 11(5): e1004248)—acomputational protein-protein interaction (PPI) method to predictinteractions mediated by domain-motif interfaces. PrePPI (PredictingPPI), a structure based PPI prediction method, combines structuralevidence with non-structural evidence using a Bayesian statisticalframework. The method involves taking a pair a query proteins and usingstructural alignment to identify structural representatives thatcorrespond to either their experimentally determined structures orhomology models. Structural alignment is further used to identify bothclose and remote structural neighbors by considering global and localgeometric relationships. Whenever two neighbors of the structuralrepresentatives form a complex reported in the Protein Data Bank, thisdefines a template for modelling the interaction between the two queryproteins. Models of the complex are created by superimposing therepresentative structures on their corresponding structural neighbor inthe template. This approach is further described in Dey et al., 2013(Prot Sci; 22: 359-66).

For purpose of this invention, amplification means any method employinga primer and a polymerase capable of replicating a target sequence withreasonable fidelity. Amplification may be carried out by natural orrecombinant DNA polymerases such as TaqGold™, T7 DNA polymerase, Klenowfragment of E. coli DNA polymerase, and reverse transcriptase. Apreferred amplification method is PCR. In certain aspects the inventioninvolves vectors. A used herein, a “vector” is a tool that allows orfacilitates the transfer of an entity from one environment to another.It is a replicon, such as a plasmid, phage, or cosmid, into whichanother DNA segment may be inserted so as to bring about the replicationof the inserted segment. Generally, a vector is capable of replicationwhen associated with the proper control elements. In general, the term“vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. Vectors include, butare not limited to, nucleic acid molecules that are single-stranded,double-stranded, or partially double-stranded; nucleic acid moleculesthat comprise one or more free ends, no free ends (e.g., circular);nucleic acid molecules that comprise DNA, RNA, or both; and othervarieties of polynucleotides known in the art. One type of vector is a“plasmid,” which refers to a circular double stranded DNA loop intowhich additional DNA segments can be inserted, such as by standardmolecular cloning techniques. Another type of vector is a viral vector,wherein virally-derived DNA or RNA sequences are present in the vectorfor packaging into a virus (e.g., retroviruses, replication defectiveretroviruses, adenoviruses, replication defective adenoviruses, andadeno-associated viruses (AAVs)). Viral vectors also includepolynucleotides carried by a virus for transfection into a host cell.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g., bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively-linked. Such vectors are referred to herein as “expressionvectors.” Common expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. Recombinant expressionvectors can comprise a nucleic acid of the invention in a form suitablefor expression of the nucleic acid in a host cell, which means that therecombinant expression vectors include one or more regulatory elements,which may be selected on the basis of the host cells to be used forexpression, that is operatively-linked to the nucleic acid sequence tobe expressed. Within a recombinant expression vector, “operably linked”is intended to mean that the nucleotide sequence of interest is linkedto the regulatory element(s) in a manner that allows for expression ofthe nucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a host cell when the vector is introduced into the hostcell). With regards to recombination and cloning methods, mention ismade of U.S. patent application Ser. No. 10/815,730, published Sep. 2,2004 as US 2004-0171156 A1, the contents of which are hereinincorporated by reference in their entirety. Aspects of the inventionrelate to bicistronic vectors for guide RNA and wild type, modified ormutated CRISPR effector proteins/enzymes (e.g. Cas13b effectorproteins). Bicistronic expression vectors guide RNA and wild type,modified or mutated CRISPR effector proteins/enzymes (e.g. Cas13beffector proteins) are preferred. In general and particularly in thisembodiment and wild type, modified or mutated CRISPR effectorproteins/enzymes (e.g. Cas13b effector proteins) is preferably driven bythe CBh promoter. The RNA may preferably be driven by a Pol IIIpromoter, such as a U6 promoter. Ideally the two are combined.

In some embodiments, a loop in the guide RNA or crRNA is provided. Thismay be a stem loop or a tetra loop. The loop is preferably GAAA, but itis not limited to this sequence or indeed to being only 4 bp in length.Indeed, preferred loop forming sequences for use in hairpin structuresare four nucleotides in length, and most preferably have the sequenceGAAA. However, longer or shorter loop sequences may be used, as mayalternative sequences. The sequences preferably include a nucleotidetriplet (for example, AAA), and an additional nucleotide (for example Cor G). Examples of loop forming sequences include CAAA and AAAG.

In practicing any of the methods disclosed herein, a suitable vector canbe introduced to a cell or an embryo via one or more methods known inthe art, including without limitation, microinjection, electroporation,sonoporation, biolistics, calcium phosphate-mediated transfection,cationic transfection, liposome transfection, dendrimer transfection,heat shock transfection, nucleofection transfection, magnetofection,lipofection, impalefection, optical transfection, proprietaryagent-enhanced uptake of nucleic acids, and delivery via liposomes,immunoliposomes, virosomes, or artificial virions. In some methods, thevector is introduced into an embryo by microinjection. The vector orvectors may be microinjected into the nucleus or the cytoplasm of theembryo. In some methods, the vector or vectors may be introduced into acell by nucleofection.

Vectors can be designed for expression of CRISPR transcripts (e.g.,nucleic acid transcripts, proteins, or enzymes) in prokaryotic oreukaryotic cells. For example, CRISPR transcripts can be expressed inbacterial cells such as Escherichia coli, insect cells (usingbaculovirus expression vectors), yeast cells, or mammalian cells.Suitable host cells are discussed further in Goeddel, GENE EXPRESSIONTECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.(1990). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Vectors may be introduced and propagated in a prokaryote or prokaryoticcell. In some embodiments, a prokaryote is used to amplify copies of avector to be introduced into a eukaryotic cell or as an intermediatevector in the production of a vector to be introduced into a eukaryoticcell (e.g., amplifying a plasmid as part of a viral vector packagingsystem). In some embodiments, a prokaryote is used to amplify copies ofa vector and express one or more nucleic acids, such as to provide asource of one or more proteins for delivery to a host cell or hostorganism. Expression of proteins in prokaryotes is most often carriedout in Escherichia coli with vectors containing constitutive orinducible promoters directing the expression of either fusion ornon-fusion proteins. Fusion vectors add a number of amino acids to aprotein encoded therein, such as to the amino terminus of therecombinant protein. Such fusion vectors may serve one or more purposes,such as: (i) to increase expression of recombinant protein; (ii) toincrease the solubility of the recombinant protein; and (iii) to aid inthe purification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase. Example fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant protein. Examples of suitableinducible non-fusion E. coli expression vectors include pTrc (Amrann etal., (1988) Gene 69:301-315) and pET 11d (Studier et al., GENEEXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, SanDiego, Calif. (1990) 60-89). In some embodiments, a vector is a yeastexpression vector. Examples of vectors for expression in yeastSaccharomyces cerivisae include pYepSecl (Baldari, et al., 1987. EMBO J.6: 229-234), pMFa (Kuijan and Herskowitz, 1982. Cell 30: 933-943),pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (InvitrogenCorporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego,Calif.). In some embodiments, a vector drives protein expression ininsect cells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., SF9cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170:31-39). In some embodiments, a vector is capable of driving expressionof one or more sequences in mammalian cells using a mammalian expressionvector. Examples of mammalian expression vectors include pCDM8 (Seed,1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO 1 6:187-195). When used in mammalian cells, the expression vector's controlfunctions are typically provided by one or more regulatory elements. Forexample, commonly used promoters are derived from polyoma, adenovirus 2,cytomegalovirus, simian virus 40, and others disclosed herein and knownin the art. For other suitable expression systems for both prokaryoticand eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al.,MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989. In some embodiments, the recombinant mammalian expressionvector is capable of directing expression of the nucleic acidpreferentially in a particular cell type (e.g., tissue-specificregulatory elements are used to express the nucleic acid).Tissue-specific regulatory elements are known in the art. Non-limitingexamples of suitable tissue-specific promoters include the albuminpromoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277),lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto andBaltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Baneiji, etal., 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter;Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477),pancreas-specific promoters (Edlund, et al., 1985. Science 230:912-916), and mammary gland-specific promoters (e.g., milk wheypromoter; U.S. Pat. No. 4,873,316 and European Application PublicationNo. 264,166). Developmentally-regulated promoters are also encompassed,e.g., the murine hox promoters (Kessel and Gruss, 1990. Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman, 1989.Genes Dev. 3: 537-546). With regards to these prokaryotic and eukaryoticvectors, mention is made of U.S. Pat. No. 6,750,059, the contents ofwhich are incorporated by reference herein in their entirety. Otherembodiments of the invention may relate to the use of viral vectors,with regards to which mention is made of U.S. patent application Ser.No. 13/092,085, the contents of which are incorporated by referenceherein in their entirety. Tissue-specific regulatory elements are knownin the art and in this regard, mention is made of U.S. Pat. No.7,776,321, the contents of which are incorporated by reference herein intheir entirety.

In some embodiments, a regulatory element is operably linked to one ormore elements of or encoding a CRISPR Cas13b system or complex so as todrive expression of the one or more elements of the CRISPR system. Ingeneral, CRISPRs (Clustered Regularly Interspaced Short PalindromicRepeats), also known as SPIDRs (SPacer Interspersed Direct Repeats),constitute a family of DNA loci that are usually specific to aparticular bacterial species. The CRISPR locus comprises a distinctclass of interspersed short sequence repeats (SSRs) that were recognizedin E. coli (Ishino et al., J. Bacteriol., 169:5429-5433 [1987]; andNakata et al., J. Bacteriol., 171:3553-3556 [1989]), and associatedgenes. Similar interspersed SSRs have been identified in Haloferaxmediterranei, Streptococcus pyogenes, Anabaena, and Mycobacteriumtuberculosis (See, Groenen et al., Mol. Microbiol., 10:1057-1065 [1993];Hoe et al., Emerg. Infect. Dis., 5:254-263 [1999]; Masepohl et al.,Biochim. Biophys. Acta 1307:26-30 [1996]; and Mojica et al., Mol.Microbiol., 17:85-93 [1995]). The CRISPR loci typically differ fromother SSRs by the structure of the repeats, which have been termed shortregularly spaced repeats (SRSRs) (Janssen et al., OMICS J. Integ. Biol.,6:23-33 [2002]; and Mojica et al., Mol. Microbiol., 36:244-246 [2000]).In general, the repeats are short elements that occur in clusters thatare regularly spaced by unique intervening sequences with asubstantially constant length (Mojica et al., [2000], supra). Althoughthe repeat sequences are highly conserved between strains, the number ofinterspersed repeats and the sequences of the spacer regions typicallydiffer from strain to strain (van Embden et al., J. Bacteriol.,182:2393-2401 [2000]). CRISPR loci have been identified in more than 40prokaryotes (See e.g., Jansen et al., Mol. Microbiol., 43:1565-1575[2002]; and Mojica et al., [2005]) including, but not limited toAeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula,Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus,Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium,Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus,Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma,Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas,Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella,Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus,Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia,Treponema, and Thermotoga.

In general, “RNA-targeting system” as used in the present applicationrefers collectively to transcripts and other elements involved in theexpression of or directing the activity of RNA-targetingCRISPR-associated 13b (“Cas13b”) genes (also referred to herein as aneffector protein), including sequences encoding a RNA-targeting Cas(effector) protein and a guide RNA (or crRNA sequence), with referenceto FIG. 1 as herein discussed. In general, a RNA-targeting system ischaracterized by elements that promote the formation of a RNA-targetingcomplex at the site of a target sequence. In the context of formation ofa RNA-targeting complex, “target sequence” refers to a RNA sequence towhich a guide sequence (or the guide or of the crRNA) is designed tohave complementarity, where hybridization between a target sequence anda guide RNA promotes the formation of a RNA-targeting complex. Fullcomplementarity is not necessarily required, provided there issufficient complementarity to cause hybridization and promote formationof a RNA-targeting complex. In some embodiments, a target sequence islocated in the nucleus or cytoplasm of a cell. In some embodiments, thetarget sequence may be within an organelle of a eukaryotic cell. Asequence or template that may be used for recombination into thetargeted locus comprising the target sequences is referred to as an“editing template” or “editing RNA” or “editing sequence”. In aspects ofthe invention, an exogenous template RNA may be referred to as anediting template. In an aspect of the invention the recombination ishomologous recombination. In general, a guide sequence is anypolynucleotide sequence having sufficient complementarity with a targetpolynucleotide sequence to hybridize with the target sequence and directsequence-specific binding of a nucleic acid-targeting complex to thetarget sequence. In some embodiments, the degree of complementaritybetween a guide sequence and its corresponding target sequence, whenoptimally aligned using a suitable alignment algorithm, is about or morethan about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.Optimal alignment may be determined with the use of any suitablealgorithm for aligning sequences, non-limiting example of which includethe Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithmsbased on the Burrows-Wheeler Transform (e.g. the Burrows WheelerAligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies,ELAND (Illumina, San Diego, Calif.), SOAP (available atsoap.genomics.org.cn), and Maq (available at maq.sourceforge.net). Insome embodiments, a guide sequence is about or more than about 5, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In someembodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30,25, 20, 15, 12, or fewer nucleotides in length. The ability of a guidesequence to direct sequence-specific binding of a RNA-targeting complexto a target sequence may be assessed by any suitable assay. A templatepolynucleotide may be of any suitable length, such as about or more thanabout 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, or morenucleotides in length. In some embodiments, the template polynucleotideis complementary to a portion of a polynucleotide comprising the targetsequence. When optimally aligned, a template polynucleotide mightoverlap with one or more nucleotides of a target sequences (e.g. aboutor more than about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80,90, 100 or more nucleotides). In some embodiments, when a templatesequence and a polynucleotide comprising a target sequence are optimallyaligned, the nearest nucleotide of the template polynucleotide is withinabout 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000,10000, or more nucleotides from the target sequence. In someembodiments, the RNA-targeting effector protein is part of a fusionprotein comprising one or more heterologous protein domains (e.g., aboutor more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains inaddition to the nucleic acid-targeting effector protein). In someembodiments, the CRISPR Cas13b effector protein/enzyme is part of afusion protein comprising one or more heterologous protein domains (e.g.about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domainsin addition to the CRISPR Cas13b enzyme). Examples of protein domainsthat may be fused to an effector protein include, without limitation,epitope tags, reporter gene sequences, and protein domains having one ormore of the following activities: methylase activity, demethylaseactivity, transcription activation activity, transcription repressionactivity, transcription release factor activity, histone modificationactivity, RNA cleavage activity and nucleic acid binding activity.Non-limiting examples of epitope tags include histidine (His) tags, V5tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-Gtags, and thioredoxin (Trx) tags. Examples of reporter genes include,but are not limited to, glutathione-S-transferase (GST), horseradishperoxidase (HRP), chloramphenicol acetyltransferase (CAT)beta-galactosidase, beta-glucuronidase, luciferase, green fluorescentprotein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellowfluorescent protein (YFP), and autofluorescent proteins including bluefluorescent protein (BFP). A nucleic acid-targeting effector protein maybe fused to a gene sequence encoding a protein or a fragment of aprotein that bind DNA molecules or bind other cellular molecules,including but not limited to maltose binding protein (MBP), S-tag, Lex ADNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, andherpes simplex virus (HSV) BP16 protein fusions. Additional domains thatmay form part of a fusion protein comprising a nucleic acid-targetingeffector protein are described in US20110059502, incorporated herein byreference. In some embodiments, a tagged nucleic acid-targeting effectorprotein is used to identify the location of a target sequence. In someembodiments, a CRISPR Cas13b enzyme may form a component of an induciblesystem. The inducible nature of the system would allow forspatiotemporal control of gene editing or gene expression using a formof energy. The form of energy may include but is not limited toelectromagnetic radiation, sound energy, chemical energy and thermalenergy. Examples of inducible system include tetracycline induciblepromoters (Tet-On or Tet-Off), small molecule two-hybrid transcriptionactivations systems (FKBP, ABA, etc), or light inducible systems(Phytochrome, LOV domains, or cryptochrome). In one embodiment, theCRISPR Cas13b enzyme may be a part of a Light Inducible TranscriptionalEffector (LITE) to direct changes in transcriptional activity in asequence-specific manner. The components of a light may include a CRISPRenzyme, a light-responsive cytochrome heterodimer (e.g. from Arabidopsisthaliana), and a transcriptional activation/repression domain. Furtherexamples of inducible DNA binding proteins and methods for their use areprovided in U.S. 61/736,465 and U.S. 61/721,283 and WO 2014/018423 andU.S. Pat. Nos. 8,889,418, 8,895,308, US20140186919, US20140242700,US20140273234, US20140335620, WO2014093635, which is hereby incorporatedby reference in its entirety. In some aspects, the invention providesmethods comprising delivering one or more polynucleotides, such as orone or more vectors as described herein, one or more transcriptsthereof, and/or one or proteins transcribed therefrom, to a host cell.In some aspects, the invention further provides cells produced by suchmethods, and organisms (such as animals, plants, or fungi) comprising orproduced from such cells. In some embodiments, a RNA-targeting effectorprotein in combination with (and optionally complexed with) a guide RNAor crRNA is delivered to a cell. Conventional viral and non-viral basedgene transfer methods can be used to introduce nucleic acids inmammalian cells or target tissues. Such methods can be used toadminister nucleic acids encoding components of a RNA-targeting systemto cells in culture, or in a host organism. Non-viral vector deliverysystems include DNA plasmids, RNA (e.g. a transcript of a vectordescribed herein), naked nucleic acid, and nucleic acid complexed with adelivery vehicle, such as a liposome. Viral vector delivery systemsinclude DNA and RNA viruses, which have either episomal or integratedgenomes after delivery to the cell. For a review of gene therapyprocedures, see Anderson, Science 256:808-813 (1992); Nabel & Felgner,TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993);Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992);Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, RestorativeNeurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, BritishMedical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topicsin Microbiology and Immunology, Doerfler and Bohm (eds) (1995); and Yuet al., Gene Therapy 1:13-26 (1994). Methods of non-viral delivery ofnucleic acids include lipofection, nucleofection, microinjection,biolistics, virosomes, liposomes, immunoliposomes, polycation orlipid:nucleic acid conjugates, naked DNA, artificial virions, andagent-enhanced uptake of DNA. Lipofection is described in e.g., U.S.Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagentsare sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic andneutral lipids that are suitable for efficient receptor-recognitionlipofection of polynucleotides include those of Felgner, WO 91/17424; WO91/16024. Delivery can be to cells (e.g. in vitro or ex vivoadministration) or target tissues (e.g. in vivo administration).

Models of Conditions

A method of the invention may be used to create a plant, an animal orcell that may be used to model and/or study genetic or epigeneticconditions of interest, such as a through a model of mutations ofinterest or a disease model. As used herein, “disease” refers to adisease, disorder, or indication in a subject. For example, a method ofthe invention may be used to create an animal or cell that comprises amodification in one or more nucleic acid sequences associated with adisease, or a plant, animal or cell in which expression of one or morenucleic acid sequences associated with a disease are altered. Such anucleic acid sequence may encode or be translated a disease associatedprotein sequence or may be a disease associated control sequence.Accordingly, it is understood that in embodiments of the invention, aplant, subject, patient, organism or cell can be a non-human subject,patient, organism or cell. Thus, the invention provides a plant, animalor cell, produced by the present methods, or a progeny thereof. Theprogeny may be a clone of the produced plant or animal, or may resultfrom sexual reproduction by crossing with other individuals of the samespecies to introgress further desirable traits into their offspring. Thecell may be in vivo or ex vivo in the cases of multicellular organisms,particularly animals or plants. In the instance where the cell is incultured, a cell line may be established if appropriate culturingconditions are met and preferably if the cell is suitably adapted forthis purpose (for instance a stem cell). Bacterial cell lines producedby the invention are also envisaged. Hence, cell lines are alsoenvisaged. In some methods, the disease model can be used to study theeffects of mutations, or more general altered, such as reduced,expression of genes or gene products on the animal or cell anddevelopment and/or progression of the disease using measures commonlyused in the study of the disease. Alternatively, such a disease model isuseful for studying the effect of a pharmaceutically active compound onthe disease. In some methods, the disease model can be used to assessthe efficacy of a potential gene therapy strategy. That is, adisease-associated RNA can be modified such that the disease developmentand/or progression is displayed or inhibited or reduced and then effectsof a compound on the progression or inhibition or reduction are tested.

Useful in the practice of the instant invention utilizing Cas13beffector proteins and complexes thereof and nucleic acid moleculesencoding same and methods using same, reference is made to: Genome-ScaleCRISPR-Cas9 Knockout Screening in Human Cells. Shalem, O., Sanjana, NE., Hartenian, E., Shi, X., Scott, D A., Mikkelson, T., Heckl, D.,Ebert, B L., Root, D E., Doench, J G., Zhang, F. Science December 12.(2013). [Epub ahead of print]; Published in final edited form as:Science. 2014 Jan. 3; 343(6166): 84-87. Shalem et al. involves a new wayto interrogate gene function on a genome-wide scale. Their studiesshowed that delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO)library targeted 18,080 genes with 64,751 unique guide sequences enabledboth negative and positive selection screening in human cells. First,the authors showed use of the GeCKO library to identify genes essentialfor cell viability in cancer and pluripotent stem cells. Next, in amelanoma model, the authors screened for genes whose loss is involved inresistance to vemurafenib, a therapeutic that inhibits mutant proteinkinase BRAF. Their studies showed that the highest-ranking candidatesincluded previously validated genes NF1 and MED12 as well as novelhitsNF2, CUL3, TADA2B, and TADA1. The authors observed a high level ofconsistency between independent guide RNAs targeting the same gene and ahigh rate of hit confirmation, and thus demonstrated the promise ofgenome-scale screening with Cas9. Reference is also made to US patentpublication number US20140357530; and PCT Patent PublicationWO2014093701, hereby incorporated herein by reference.

The term “associated with” is used here in relation to the associationof the functional domain to the Cas13b effector protein or the adaptorprotein. It is used in respect of how one molecule ‘associates’ withrespect to another, for example between an adaptor protein and afunctional domain, or between the Cas13b effector protein and afunctional domain. In the case of such protein-protein interactions,this association may be viewed in terms of recognition in the way anantibody recognizes an epitope. Alternatively, one protein may beassociated with another protein via a fusion of the two, for instanceone subunit being fused to another subunit. Fusion typically occurs byaddition of the amino acid sequence of one to that of the other, forinstance via splicing together of the nucleotide sequences that encodeeach protein or subunit. Alternatively, this may essentially be viewedas binding between two molecules or direct linkage, such as a fusionprotein. In any event, the fusion protein may include a linker betweenthe two subunits of interest (i.e. between the enzyme and the functionaldomain or between the adaptor protein and the functional domain). Thus,in some embodiments, the Cas13b effector protein or adaptor protein isassociated with a functional domain by binding thereto. In otherembodiments, the Cas13b effector protein or adaptor protein isassociated with a functional domain because the two are fused together,optionally via an intermediate linker.

Cas13b Effector Protein Complexes can be Used in Plants

The invention in some embodiments comprehends a method of modifying ancell or organism. The cell may be a prokaryotic cell or a eukaryoticcell. The cell may be a mammalian cell. The mammalian cell many be anon-human primate, bovine, porcine, rodent or mouse cell. The cell maybe a non-mammalian eukaryotic cell such as poultry, fish or shrimp. Thecell may also be a plant cell. The plant cell may be of a crop plantsuch as cassava, corn, sorghum, wheat, or rice. The plant cell may alsobe of an algae, tree or vegetable. The modification introduced to thecell by the present invention may be such that the cell and progeny ofthe cell are altered for improved production of biologic products suchas an antibody, starch, alcohol or other desired cellular output. Themodification introduced to the cell by the present invention may be suchthat the cell and progeny of the cell include an alteration that changesthe biologic product produced. The system may comprise one or moredifferent vectors. In an aspect of the invention, the effector proteinis codon optimized for expression the desired cell type, preferentiallya eukaryotic cell, preferably a mammalian cell or a human cell. Cas13bsystem(s) (e.g., single or multiplexed) can be used in conjunction withrecent advances in crop genomics. Such CRISPR system(s) can be used toperform efficient and cost effective plant gene or genome ortranscriptome interrogation or editing or manipulation—for instance, forrapid investigation and/or selection and/or interrogations and/orcomparison and/or manipulations and/or transformation of plant genes orgenomes; e.g., to create, identify, develop, optimize, or confertrait(s) or characteristic(s) to plant(s) or to transform a plantgenome. There can accordingly be improved production of plants, newplants with new combinations of traits or characteristics or new plantswith enhanced traits. Such CRISPR system(s) can be used with regard toplants in Site-Directed Integration (SDI) or Gene Editing (GE) or anyNear Reverse Breeding (NRB) or Reverse Breeding (RB) techniques.Accordingly, reference herein to animal cells may also apply, mutatismutandis, to plant cells unless otherwise apparent; and, the enzymesherein having reduced off-target effects and systems employing suchenzymes can be used in plant applications, including those mentionedherein. Engineered plants modified by the effector protein (Cas13b) andsuitable guide (crRNA), and progeny thereof, as provided. These mayinclude disease or drought resistant crops, such as wheat, barley, rice,soybean or corn; plants modified to remove or reduce the ability toself-pollinate (but which can instead, optionally, hybridise instead);and allergenic foods such as peanuts and nuts where the immunogenicproteins have been disabled, destroyed or disrupted by targeting via aeffector protein and suitable guide. Any aspect of using classicalCRIPSR-Cas systems may be adapted to use in CRISPR systems that are Casprotein agnostic, e.g. Cas13b effector protein systems.

Therapeutic Treatment

The system of the invention can be applied in areas of former RNAcutting technologies, without undue experimentation, from thisdisclosure, including therapeutic, assay and other applications, becausethe present application provides the foundation for informed engineeringof the system. The present invention provides for therapeutic treatmentof a disease caused by overexpression of RNA, toxic RNA and/or mutatedRNA (such as, for example, splicing defects or truncations). Expressionof the toxic RNA may be associated with formation of nuclear inclusionsand late-onset degenerative changes in brain, heart or skeletal muscle.In the best studied example, myotonic dystrophy, it appears that themain pathogenic effect of the toxic RNA is to sequester binding proteinsand compromise the regulation of alternative splicing (Hum. Mol. Genet.(2006) 15 (suppl 2): R162-R169). Myotonic dystrophy [dystrophiamyotonica (DM)] is of particular interest to geneticists because itproduces an extremely wide range of clinical features. A partial listingwould include muscle wasting, cataracts, insulin resistance, testicularatrophy, slowing of cardiac conduction, cutaneous tumors and effects oncognition. The classical form of DM, which is now called DM type 1(DM1), is caused by an expansion of CTG repeats in the 3′-untranslatedregion (UTR) of DMPK, a gene encoding a cytosolic protein kinase.

The innate immune system detects viral infection primarily byrecognizing viral nucleic acids inside an infected cell, referred to asDNA or RNA sensing. In vitro RNA sensing assays can be used to detectspecific RNA substrates. The RNA targeting effector protein can forinstance be used for RNA-based sensing in living cells. Examples ofapplications are diagnostics by sensing of, for examples,disease-specific RNAs. The RNA targeting effector protein (Cas13b) ofthe invention can further be used for antiviral activity, in particularagainst RNA viruses. The effector protein (Cas13b) can be targeted tothe viral RNA using a suitable guide RNA selective for a selected viralRNA sequence. In particular, the effector protein may be an activenuclease that cleaves RNA, such as single stranded RNA. Therapeuticdosages of the enzyme system of the present invention to target RNA theabove-referenced RNAs are contemplated to be about 0.1 to about 2 mg/kgthe dosages may be administered sequentially with a monitored response,and repeated dosages if necessary, up to about 7 to 10 doses perpatient. Advantageously, samples are collected from each patient duringthe treatment regimen to ascertain the effectiveness of treatment. Forexample, RNA samples may be isolated and quantified to determine ifexpression is reduced or ameliorated. Such a diagnostic is within thepurview of one of skill in the art.

Transcriptome Wide Knock-Down Screening

The CRISPR effector protein complexes described herein can be used toperform efficient and cost effective functional transcriptonic screens.Such screens can utilize CRISPR effector protein based transcriptomewide libraries. Such screens and libraries can provide for determiningthe function of genes, cellular pathways genes are involved in, and howany alteration in gene expression can result in a particular biologicalprocess. An advantage of the present invention is that the CRISPR systemavoids off-target binding and its resulting side effects. This isachieved using systems arranged to have a high degree of sequencespecificity for the target RNA. In preferred embodiments of theinvention, the CRISPR effector protein complexes are Cas13b effectorprotein complexes.

In embodiments of the invention, a transcriptome wide library maycomprise a plurality of Cas13b guide RNAs, as described herein,comprising guide sequences that are capable of targeting a plurality oftarget sequences in a plurality of loci in a population of eukaryoticcells. The population of cells may be a population of embryonic stem(ES) cells. The target sequence in the locus may be a non-codingsequence. The non-coding sequence may be an intron, regulatory sequence,splice site, 3′ UTR, 5′ UTR, or polyadenylation signal. Gene function ofone or more gene products may be altered by said targeting. Thetargeting may result in a knockout of gene function. The targeting of agene product may comprise more than one guide RNA. A gene product may betargeted by 2, 3, 4, 5, 6, 7, 8, 9, or 10 guide RNAs, preferably 3 to 4per gene. Off-target modifications may be minimized by exploiting thestaggered double strand breaks generated by Cas13b effector proteincomplexes or by utilizing methods analogous to those used in CRISPR-Cas9systems (See, e.g., DNA targeting specificity of RNA-guided Cas9nucleases. Hsu, P., Scott, D., Weinstein, J., Ran, F A., Konermann, S.,Agarwala, V., Li, Y., Fine, E., Wu, X., Shalem, O., Cradick, T J.,Marraffini, L A., Bao, Zhang, F. Nat Biotechnol doi:10.1038/nbt.2647(2013)), incorporated herein by reference. The targeting may be of about100 or more sequences. The targeting may be of about 1000 or moresequences. The targeting may be of about 20,000 or more sequences. Thetargeting may be of the entire genome. The targeting may be of a panelof target sequences focused on a relevant or desirable pathway. Thepathway may be an immune pathway. The pathway may be a cell divisionpathway.

One aspect of the invention comprehends a transcriptome wide librarythat may comprise a plurality of cas13b guide RNAs that may compriseguide sequences that are capable of targeting a plurality of targetsequences in a plurality of loci, wherein said targeting results in aknockdown of gene function. This library may potentially comprise guideRNAs that target each and every gene in the genome of an organism.

In some embodiments of the invention the organism or subject is aeukaryote (including mammal including human) or a non-human eukaryote ora non-human animal or a non-human mammal. In some embodiments, theorganism or subject is a non-human animal, and may be an arthropod, forexample, an insect, or may be a nematode. In some methods of theinvention the organism or subject is a plant. In some methods of theinvention the organism or subject is a mammal or a non-human mammal. Anon-human mammal may be for example a rodent (preferably a mouse or arat), an ungulate, or a primate. In some methods of the invention theorganism or subject is algae, including microalgae, or is a fungus.

The knockdown of gene function may comprise: introducing into each cellin the population of cells a vector system of one or more vectorscomprising an engineered, non-naturally occurring Cas13b effectorprotein system comprising I. a Cas13b effector protein, and II. one ormore guide RNAs, wherein components I and II may be same or on differentvectors of the system, integrating components I and II into each cell,wherein the guide sequence targets a unique gene in each cell, whereinthe Cas13b effector protein is operably linked to a regulatory element,wherein when transcribed, the guide RNA comprising the guide sequencedirects sequence-specific binding of the Cas13b effector protein systemto a target sequence in the genomic loci of the unique gene, inducingcleavage of the genomic loci by the Cas13b effector protein, andconfirming different knockdown events in a plurality of unique genes ineach cell of the population of cells thereby generating a gene knockdowncell library. The invention comprehends that the population of cells isa population of eukaryotic cells, and in a preferred embodiment, thepopulation of cells is a population of embryonic stem (ES) cells.

The one or more vectors may be plasmid vectors. The vector may be asingle vector comprising a Cas13b effector protein, a sgRNA, andoptionally, a selection marker into target cells. Not being bound by atheory, the ability to simultaneously deliver a Cas13b effector proteinand sgRNA through a single vector enables application to any cell typeof interest, without the need to first generate cell lines that expressthe Cas13b effector protein. The regulatory element may be an induciblepromoter. The inducible promoter may be a doxycycline induciblepromoter. In some methods of the invention the expression of the guidesequence is under the control of the T7 promoter and is driven by theexpression of T7 polymerase. The confirming of different knockdownevents may be by whole transcriptome sequencing. The knockdown event maybe achieved in 100 or more unique genes. The knockdown event may beachieved in 1000 or more unique genes. The knockdown event may beachieved in 20,000 or more unique genes. The knockdown event may beachieved in the entire transcriptome. The knockdown of gene function maybe achieved in a plurality of unique genes which function in aparticular physiological pathway or condition. The pathway or conditionmay be an immune pathway or condition. The pathway or condition may be acell division pathway or condition.

The invention also provides kits that comprise the transcriptome widelibraries mentioned herein. The kit may comprise a single containercomprising vectors or plasmids comprising the library of the invention.The kit may also comprise a panel comprising a selection of uniqueCas13b effector protein system guide RNAs comprising guide sequencesfrom the library of the invention, wherein the selection is indicativeof a particular physiological condition. The invention comprehends thatthe targeting is of about 100 or more sequences, about 1000 or moresequences or about 20,000 or more sequences or the entire transcriptome.Furthermore, a panel of target sequences may be focused on a relevant ordesirable pathway, such as an immune pathway or cell division.

In an additional aspect of the invention, the Cas13b effector proteinmay comprise one or more mutations and may be used as a generic RNAbinding protein with or without fusion to a functional domain. Themutations may be artificially introduced mutations or gain- orloss-of-function mutations. The mutations have been characterized asdescribed herein. In one aspect of the invention, the functional domainmay be a transcriptional activation domain, which may be VP64. In otheraspects of the invention, the functional domain may be a transcriptionalrepressor domain, which may be KRAB or SID4×. Other aspects of theinvention relate to the mutated Cas13b effector protein being fused todomains which include but are not limited to a transcriptionalactivator, repressor, a recombinase, a transposase, a histone remodeler,a demethylase, a DNA methyltransferase, a cryptochrome, a lightinducible/controllable domain or a chemically inducible/controllabledomain. Some methods of the invention can include inducing expression oftargeted genes. In one embodiment, inducing expression by targeting aplurality of target sequences in a plurality of genomic loci in apopulation of eukaryotic cells is by use of a functional domain.

Useful in the practice of the instant invention utilizing Cas13beffector protein complexes are methods used in CRISPR-Cas9 systems andreference is made to:

Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Shalem, O.,Sanjana, N E., Hartenian, E., Shi, X., Scott, D A., Mikkelson, T.,Heckl, D., Ebert, B L., Root, D E., Doench, J G., Zhang, F. Science Dec.12, 2013. [Epub ahead of print]; Published in final edited form as:Science. 2014 Jan. 3; 343(6166): 84-87.

Shalem et al. involves a new way to interrogate gene function on agenome-wide scale. Their studies showed that delivery of a genome-scaleCRISPR-Cas9 knockout (GeCKO) library targeted 18,080 genes with 64,751unique guide sequences enabled both negative and positive selectionscreening in human cells. First, the authors showed use of the GeCKOlibrary to identify genes essential for cell viability in cancer andpluripotent stem cells. Next, in a melanoma model, the authors screenedfor genes whose loss is involved in resistance to vemurafenib, atherapeutic that inhibits mutant protein kinase BRAF. Their studiesshowed that the highest-ranking candidates included previously validatedgenes NF1 and MED12 as well as novel hitsNF2, CUL3, TADA2B, and TADA1.The authors observed a high level of consistency between independentguide RNAs targeting the same gene and a high rate of hit confirmation,and thus demonstrated the promise of genome-scale screening with Cas9.

Reference is also made to US patent publication number US20140357530;and PCT Patent Publication WO2014093701, hereby incorporated herein byreference.

Functional Alteration and Screening

In another aspect, the present invention provides for a method offunctional evaluation and screening of genes. The use of the CRISPRsystem of the present invention to precisely deliver functional domains,to activate or repress genes or to alter epigenetic state by preciselyaltering the methylation site on a a specific locus of interest, can bewith one or more guide RNAs applied to a single cell or population ofcells or with a library applied to genome in a pool of cells ex vivo orin vivo comprising the administration or expression of a librarycomprising a plurality of guide RNAs (sgRNAs) and wherein the screeningfurther comprises use of a Cas13b effector protein, wherein the CRISPRcomplex comprising the Cas13b effector protein is modified to comprise aheterologous functional domain. In an aspect the invention provides amethod for screening a genome/transcriptome comprising theadministration to a host or expression in a host in vivo of a library.In an aspect the invention provides a method as herein discussed furthercomprising an activator administered to the host or expressed in thehost. In an aspect the invention provides a method as herein discussedwherein the activator is attached to a Cas13b effector protein. In anaspect the invention provides a method as herein discussed wherein theactivator is attached to the N terminus or the C terminus of the Cas13beffector protein. In an aspect the invention provides a method as hereindiscussed wherein the activator is attached to a sgRNA loop. In anaspect the invention provides a method as herein discussed furthercomprising a repressor administered to the host or expressed in thehost. In an aspect the invention provides a method as herein discussed,wherein the screening comprises affecting and detecting gene activation,gene inhibition, or cleavage in the locus.

In an aspect, the invention provides efficient on-target activity andminimizes off target activity. In an aspect, the invention providesefficient on-target cleavage by Cas13b effector protein and minimizesoff-target cleavage by the Cas13b effector protein. In an aspect, theinvention provides guide specific binding of Cas13b effector protein ata gene locus without DNA cleavage. Accordingly, in an aspect, theinvention provides target-specific gene regulation. In an aspect, theinvention provides guide specific binding of Cas13b effector protein ata gene locus without DNA cleavage. Accordingly, in an aspect, theinvention provides for cleavage at one locus and gene regulation at adifferent locus using a single Cas13b effector protein. In an aspect,the invention provides orthogonal activation and/or inhibition and/orcleavage of multiple targets using one or more Cas13b effector proteinand/or enzyme.

In an aspect the invention provides a method as herein discussed,wherein the host is a eukaryotic cell. In an aspect the inventionprovides a method as herein discussed, wherein the host is a mammaliancell. In an aspect the invention provides a method as herein discussed,wherein the host is a non-human eukaryote. In an aspect the inventionprovides a method as herein discussed, wherein the non-human eukaryoteis a non-human mammal. In an aspect the invention provides a method asherein discussed, wherein the non-human mammal is a mouse. An aspect theinvention provides a method as herein discussed comprising the deliveryof the Cas13b effector protein complexes or component(s) thereof ornucleic acid molecule(s) coding therefor, wherein said nucleic acidmolecule(s) are operatively linked to regulatory sequence(s) andexpressed in vivo. In an aspect the invention provides a method asherein discussed wherein the expressing in vivo is via a lentivirus, anadenovirus, or an AAV. In an aspect the invention provides a method asherein discussed wherein the delivery is via a particle, a nanoparticle,a lipid or a cell penetrating peptide (CPP).

In an aspect the invention provides a pair of CRISPR complexescomprising Cas13b effector protein, each comprising a guide RNA (sgRNA)comprising a guide sequence capable of hybridizing to a target sequencein a genomic locus of interest in a cell, wherein at least one loop ofeach sgRNA is modified by the insertion of distinct RNA sequence(s) thatbind to one or more adaptor proteins, and wherein the adaptor protein isassociated with one or more functional domains, wherein each sgRNA ofeach Cas13b effector protein complex comprises a functional domainhaving a DNA cleavage activity.

In an aspect the invention provides a method for cutting a targetsequence in a locus of interest comprising delivery to a cell of theCas13b effector protein complexes or component(s) thereof or nucleicacid molecule(s) coding therefor, wherein said nucleic acid molecule(s)are operatively linked to regulatory sequence(s) and expressed in vivo.In an aspect the invention provides a method as herein-discussed whereinthe delivery is via a lentivirus, an adenovirus, or an AAV.

In an aspect the invention provides a library, method or complex asherein-discussed wherein the sgRNA is modified to have at least onenon-coding functional loop, e.g., wherein the at least one non-codingfunctional loop is repressive; for instance, wherein the at least onenon-coding functional loop comprises Alu.

In one aspect, the invention provides a method for altering or modifyingexpression of a gene product. The said method may comprise introducinginto a cell containing and expressing a DNA molecule encoding the geneproduct an engineered, non-naturally occurring CRISPR system comprisinga Cas13b effector protein and guide RNA that targets the RNA molecule,whereby the guide RNA targets the RNA target molecule encoding the geneproduct and the Cas13b effector protein cleaves the RNA moleculeencoding the gene product, whereby expression of the gene product isaltered; and, wherein the Cas13b effector protein and the guide RNA donot naturally occur together. The invention comprehends the guide RNAcomprising a guide sequence linked to a direct repeat sequence. Theinvention further comprehends the Cas13b effector protein being codonoptimized for expression in a Eukaryotic cell. In a preferred embodimentthe Eukaryotic cell is a mammalian cell and in a more preferredembodiment the mammalian cell is a human cell. In a further embodimentof the invention, the expression of the gene product is decreased.

In some embodiments, one or more functional domains are associated withthe Cas13b effector protein. In some embodiments, one or more functionaldomains are associated with an adaptor protein, for example as used withthe modified guides of Konnerman et al. (Nature 517, 583-588, 29 Jan.2015). In some embodiments, one or more functional domains areassociated with an dead sgRNA (dRNA). In some embodiments, a dRNAcomplex with active Cas13b effector protein directs gene regulation by afunctional domain at on gene locus while an sgRNA directs DNA cleavageby the active Cas13b effector protein at another locus, for example asdescribed analogously in CRISPR-Cas9 systems by Dahlman et al.,‘Orthogonal gene control with a catalytically active Cas9 nuclease,’Nature Biotechnology 33, p. 1159-61 (November, 2015). In someembodiments, dRNAs are selected to maximize selectivity of regulationfor a gene locus of interest compared to off-target regulation. In someembodiments, dRNAs are selected to maximize target gene regulation andminimize target cleavage

For the purposes of the following discussion, reference to a functionaldomain could be a functional domain associated with the Cas13b effectorprotein or a functional domain associated with the adaptor protein.

In some embodiments, the one or more functional domains is an NLS(Nuclear Localization Sequence) or an NES (Nuclear Export Signal). Insome embodiments, the one or more functional domains is atranscriptional activation domain comprises VP64, p65, MyoD1, HSF1, RTA,SETT/9 and a histone acetyltransferase. Other references herein toactivation (or activator) domains in respect of those associated withthe CRISPR enzyme include any known transcriptional activation domainand specifically VP64, p65, MyoD1, HSF1, RTA, SETT/9 or a histoneacetyltransferase.

In some embodiments, the one or more functional domains is atranscriptional repressor domain. In some embodiments, thetranscriptional repressor domain is a KRAB domain. In some embodiments,the transcriptional repressor domain is a NuE domain, NcoR domain, SIDdomain or a SID4× domain.

In some embodiments, the one or more functional domains have one or moreactivities comprising translation activation activity, translationrepression activity, methylase activity, demethylase activity,transcription activation activity, transcription repression activity,transcription release factor activity, histone modification activity,RNA cleavage activity, DNA cleavage activity, DNA integration activityor nucleic acid binding activity.

In some embodiments, the DNA cleavage activity is due to a nuclease. Insome embodiments, the nuclease comprises a Fok1 nuclease. See, “DimericCRISPR RNA-guided Fok1 nucleases for highly specific genome editing”,Shengdar Q. Tsai, Nicolas Wyvekens, Cyd Khayter, Jennifer A. Foden,Vishal Thapar, Deepak Reyon, Mathew J. Goodwin, Martin J. Aryee, J.Keith Joung Nature Biotechnology 32(6): 569-77 (2014), relates todimeric RNA-guided Fok1 Nucleases that recognize extended sequences andcan edit endogenous genes with high efficiencies in human cells.

In some embodiments, the one or more functional domains is attached tothe Cas13b effector protein so that upon binding to the sgRNA and targetthe functional domain is in a spatial orientation allowing for thefunctional domain to function in its attributed function.

In some embodiments, the one or more functional domains is attached tothe adaptor protein so that upon binding of the Cas13b effector proteinto the sgRNA and target, the functional domain is in a spatialorientation allowing for the functional domain to function in itsattributed function.

In an aspect the invention provides a composition as herein discussedwherein the one or more functional domains is attached to the Cas13beffector protein or adaptor protein via a linker, optionally a GlySerlinker, as discussed herein.

It is also preferred to target endogenous (regulatory) control elements,such as involved in translation, stability, etc. Targeting of knowncontrol elements can be used to activate or repress the gene ofinterest. Targeting of putative control elements on the other hand canbe used as a means to verify such elements (by measuring the translationof the gene of interest) or to detect novel control elements (Inaddition, targeting of putative control elements can be useful in thecontext of understanding genetic causes of disease. Many mutations andcommon SNP variants associated with disease phenotypes are locatedoutside coding regions. Targeting of such regions with either theactivation or repression systems described herein can be followed byreadout of transcription of either a) a set of putative targets (e.g. aset of genes located in closest proximity to the control element) or b)whole-transcriptome readout by e.g. RNAseq or microarray. This wouldallow for the identification of likely candidate genes involved in thedisease phenotype. Such candidate genes could be useful as novel drugtargets.

Histone acetyltransferase (HAT) inhibitors are mentioned herein.However, an alternative in some embodiments is for the one or morefunctional domains to comprise an acetyltransferase, preferably ahistone acetyltransferase. These are useful in the field of epigenomics,for example in methods of interrogating the epigenome. Methods ofinterrogating the epigenome may include, for example, targetingepigenomic sequences. Targeting epigenomic sequences may include theguide being directed to an epigenomic target sequence. Epigenomic targetsequence may include, in some embodiments, include a promoter, silenceror an enhancer sequence.

Use of a functional domain linked to a Cas13b effector protein asdescribed herein, preferably a dead-Cas13b effector protein, morepreferably a dead-FnCas13b effector protein, to target epigenomicsequences can be used to activate or repress promoters, silencer orenhancers.

Examples of acetyltransferases are known but may include, in someembodiments, histone acetyltransferases. In some embodiments, thehistone acetyltransferase may comprise the catalytic core of the humanacetyltransferase p300 (Gerbasch & Reddy, Nature Biotech 6 Apr. 2015).

In some preferred embodiments, the functional domain is linked to adead-Cas13b effector protein to target and activate epigenomic sequencessuch as promoters or enhancers. One or more guides directed to suchpromoters or enhancers may also be provided to direct the binding of theCRISPR enzyme to such promoters or enhancers.

In certain embodiments, the RNA targeting effector protein of theinvention can be used to interfere with co-transcriptional modificationsof DNA/chromatin structure, RNA-directed DNA methylation, orRNA-directed silencing/activation of DNA/chromatin. RNA-directed DNAmethylation (RdDM) is an epigenetic process first discovered in plants.During RdDM, double-stranded RNAs (dsRNAs) are processed to 21-24nucleotide small interfering RNAs (siRNAs) and guide methylation ofhomologous DNA loci. Besides RNA molecules, a plethora of proteins areinvolved in the establishment of RdDM, like Argonautes, DNAmethyltransferases, chromatin remodelling complexes and theplant-specific PolIV and PolV. All these act in concert to add amethyl-group at the 5′ position of cytosines. Small RNAs can modify thechromatin structure and silence transcription by guidingArgonaute-containing complexes to complementary nascent (non-coding) RNAtrancripts. Subsequently the recruitment of chromatin-modifyingcomplexes, including histone and DNA methyltransferases, is mediated.The RNA targeting effector protein of the invention may be used totarget such small RNAs and interfere in interactions between these smallRNAs and the nascent non-coding transcripts.

The term “associated with” is used here in relation to the associationof the functional domain to the Cas13b effector protein or the adaptorprotein. It is used in respect of how one molecule ‘associates’ withrespect to another, for example between an adaptor protein and afunctional domain, or between the Cas13b effector protein and afunctional domain. In the case of such protein-protein interactions,this association may be viewed in terms of recognition in the way anantibody recognizes an epitope. Alternatively, one protein may beassociated with another protein via a fusion of the two, for instanceone subunit being fused to another subunit. Fusion typically occurs byaddition of the amino acid sequence of one to that of the other, forinstance via splicing together of the nucleotide sequences that encodeeach protein or subunit. Alternatively, this may essentially be viewedas binding between two molecules or direct linkage, such as a fusionprotein. In any event, the fusion protein may include a linker betweenthe two subunits of interest (i.e. between the enzyme and the functionaldomain or between the adaptor protein and the functional domain). Thus,in some embodiments, the Cas13b effector protein or adaptor protein isassociated with a functional domain by binding thereto. In otherembodiments, the Cas13b effector protein or adaptor protein isassociated with a functional domain because the two are fused together,optionally via an intermediate linker.

Saturating Mutagenesis

The Cas13b effector protein system(s) described herein can be used toperform saturating or deep scanning mutagenesis of genomic loci inconjunction with a cellular phenotype—for instance, for determiningcritical minimal features and discrete vulnerabilities of functionalelements required for gene expression, drug resistance, and reversal ofdisease. By saturating or deep scanning mutagenesis is meant that everyor essentially every RNA base is cut within the genomic loci. A libraryof Cas13b effector protein guide RNAs may be introduced into apopulation of cells. The library may be introduced, such that each cellreceives a single guide RNA (sgRNA). In the case where the library isintroduced by transduction of a viral vector, as described herein, a lowmultiplicity of infection (MOI) is used. The library may include sgRNAstargeting every sequence upstream of a (protospacer adjacent motif)(PAM) sequence in a genomic locus. The library may include at least 100non-overlapping genomic sequences upstream of a PAM sequence for every1000 base pairs within the genomic locus. The library may include sgRNAstargeting sequences upstream of at least one different PAM sequence. TheCas13b effector protein systems may include more than one Cas13bprotein. Any Cas13b effector protein as described herein, includingorthologues or engineered Cas13b effector proteins that recognizedifferent PAM sequences may be used. The frequency of off target sitesfor a sgRNA may be less than 500. Off target scores may be generated toselect sgRNAs with the lowest off target sites. Any phenotype determinedto be associated with cutting at a sgRNA target site may be confirmed byusing sgRNAs targeting the same site in a single experiment. Validationof a target site may also be performed by using a modified Cas13beffector protein, as described herein, and two sgRNAs targeting thegenomic site of interest. Not being bound by a theory, a target site isa true hit if the change in phenotype is observed in validationexperiments.

The Cas13b effector protein system(s) for saturating or deep scanningmutagenesis can be used in a population of cells. The Cas13b effectorprotein system(s) can be used in eukaryotic cells, including but notlimited to mammalian and plant cells. The population of cells may beprokaryotic cells. The population of eukaryotic cells may be apopulation of embryonic stem (ES) cells, neuronal cells, epithelialcells, immune cells, endocrine cells, muscle cells, erythrocytes,lymphocytes, plant cells, or yeast cells.

In one aspect, the present invention provides for a method of screeningfor functional elements associated with a change in a phenotype. Thelibrary may be introduced into a population of cells that are adapted tocontain a Cas13b effector protein. The cells may be sorted into at leasttwo groups based on the phenotype. The phenotype may be expression of agene, cell growth, or cell viability. The relative representation of theguide RNAs present in each group are determined, whereby genomic sitesassociated with the change in phenotype are determined by therepresentation of guide RNAs present in each group. The change inphenotype may be a change in expression of a gene of interest. The geneof interest may be upregulated, downregulated, or knocked out. The cellsmay be sorted into a high expression group and a low expression group.The population of cells may include a reporter construct that is used todetermine the phenotype. The reporter construct may include a detectablemarker. Cells may be sorted by use of the detectable marker.

In another aspect, the present invention provides for a method ofscreening for loci associated with resistance to a chemical compound.The chemical compound may be a drug or pesticide. The library may beintroduced into a population of cells that are adapted to contain aCas13b effector protein, wherein each cell of the population contains nomore than one guide RNA; the population of cells are treated with thechemical compound; and the representation of guide RNAs are determinedafter treatment with the chemical compound at a later time point ascompared to an early time point, whereby genomic sites associated withresistance to the chemical compound are determined by enrichment ofguide RNAs. Representation of sgRNAs may be determined by deepsequencing methods.

Useful in the practice of the instant invention utilizing Cas13beffectorprotein complexes are methods used in CRISPR-Cas9 systems and referenceis made to the article entitled BCL11A enhancer dissection byCas9-mediated in situ saturating mutagenesis. Canver, M. C., Smith, E.C., Sher, F., Pinello, L., Sanjana, N. E., Shalem, O., Chen, D. D.,Schupp, P. G., Vinjamur, D. S., Garcia, S. P., Luc, S., Kurita, R.,Nakamura, Y., Fujiwara, Y., Maeda, T., Yuan, G., Zhang, F., Orkin, S.H., & Bauer, D. E. DOI:10.1038/nature15521, published online Sep. 16,2015, the article is herein incorporated by reference and discussedbriefly below:

Canver et al. involves novel pooled CRISPR-Cas9 guide RNA libraries toperform in situ saturating mutagenesis of the human and mouse BCL11Aerythroid enhancers previously identified as an enhancer associated withfetal hemoglobin (HbF) level and whose mouse ortholog is necessary forerythroid BCL11A expression. This approach revealed critical minimalfeatures and discrete vulnerabilities of these enhancers. Throughediting of primary human progenitors and mouse transgenesis, the authorsvalidated the BCL11A erythroid enhancer as a target for HbF reinduction.The authors generated a detailed enhancer map that informs therapeuticgenome editing.

Method of Using Cas13b Systems to Modify a Cell or Organism

The invention in some embodiments comprehends a method of modifying acell or organism. The cell may be a prokaryotic cell or a eukaryoticcell. The cell may be a mammalian cell. The mammalian cell many be anon-human primate, bovine, porcine, rodent or mouse cell. The cell maybe a non-mammalian eukaryotic cell such as poultry, fish or shrimp. Thecell may also be a plant cell. The plant cell may be of a crop plantsuch as cassava, corn, sorghum, wheat, or rice. The plant cell may alsobe of an algae, tree or vegetable. The modification introduced to thecell by the present invention may be such that the cell and progeny ofthe cell are altered for improved production of biologic products suchas an antibody, starch, alcohol or other desired cellular output. Themodification introduced to the cell by the present invention may be suchthat the cell and progeny of the cell include an alteration that changesthe biologic product produced.

The system may comprise one or more different vectors. In an aspect ofthe invention, the effector protein is codon optimized for expressionthe desired cell type, preferentially a eukaryotic cell, preferably amammalian cell or a human cell.

Packaging cells are typically used to form virus particles that arecapable of infecting a host cell. Such cells include 293 cells, whichpackage adenovirus, and w2 cells or PA317 cells, which packageretrovirus. Viral vectors used in gene therapy are usually generated byproducing a cell line that packages a nucleic acid vector into a viralparticle. The vectors typically contain the minimal viral sequencesrequired for packaging and subsequent integration into a host, otherviral sequences being replaced by an expression cassette for thepolynucleotide(s) to be expressed. The missing viral functions aretypically supplied in trans by the packaging cell line. For example, AAVvectors used in gene therapy typically only possess ITR sequences fromthe AAV genome which are required for packaging and integration into thehost genome. Viral DNA is packaged in a cell line, which contains ahelper plasmid encoding the other AAV genes, namely rep and cap, butlacking ITR sequences. The cell line may also be infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV. Additionalmethods for the delivery of nucleic acids to cells are known to thoseskilled in the art. See, for example, US20030087817, incorporated hereinby reference.

In some embodiments, a host cell is transiently or non-transientlytransfected with one or more vectors described herein. In someembodiments, a cell is transfected as it naturally occurs in a subject.In some embodiments, a cell that is transfected is taken from a subject.In some embodiments, the cell is derived from cells taken from asubject, such as a cell line. A wide variety of cell lines for tissueculture are known in the art. Examples of cell lines include, but arenot limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa—S3, Huh1,Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1,CTLL-2, C1R, Rath, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480,SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55,Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E,MRCS, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS—C-1monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss,3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T,3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549,ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3,C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T,CHO Dhfr −/−, COR-L23, COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7,COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3,EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa,Hepalc1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812,KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231,MDA-MB-468, MDA-MB-435, MDCK II, MDCK II, MOR/0.2R, MONO-MAC 6, MTD-1A,MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3,NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F,RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line,U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, andtransgenic varieties thereof. Cell lines are available from a variety ofsources known to those with skill in the art (see, e.g., the AmericanType Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, acell transfected with one or more vectors described herein is used toestablish a new cell line comprising one or more vector-derivedsequences. In some embodiments, a cell transiently transfected with thecomponents of a nucleic acid-targeting system as described herein (suchas by transient transfection of one or more vectors, or transfectionwith RNA), and modified through the activity of a nucleic acid-targetingcomplex, is used to establish a new cell line comprising cellscontaining the modification but lacking any other exogenous sequence. Insome embodiments, cells transiently or non-transiently transfected withone or more vectors described herein, or cell lines derived from suchcells are used in assessing one or more test compounds.

In some embodiments, one or more vectors described herein are used toproduce a non-human transgenic animal or transgenic plant. In someembodiments, the transgenic animal is a mammal, such as a mouse, rat, orrabbit. In certain embodiments, the organism or subject is a plant. Incertain embodiments, the organism or subject or plant is algae. Methodsfor producing transgenic plants and animals are known in the art, andgenerally begin with a method of cell transfection, such as describedherein.

In one aspect, the invention provides for methods of modifying a targetpolynucleotide in a eukaryotic cell. In some embodiments, the methodcomprises allowing a nucleic acid-targeting complex to bind to thetarget polynucleotide to effect cleavage of said target polynucleotidethereby modifying the target polynucleotide, wherein the nucleicacid-targeting complex comprises a nucleic acid-targeting effectorprotein complexed with a guide RNA hybridized to a target sequencewithin said target polynucleotide.

In one aspect, the invention provides a method of modifying expressionof a polynucleotide in a eukaryotic cell. In some embodiments, themethod comprises allowing a nucleic acid-targeting complex to bind tothe polynucleotide such that said binding results in increased ordecreased expression of said polynucleotide; wherein the nucleicacid-targeting complex comprises a nucleic acid-targeting effectorprotein complexed with a guide RNA hybridized to a target sequencewithin said polynucleotide.

Cas13b Effector Protein Complexes can be Used in Plants

The Cas13b effector protein system(s) (e.g., single or multiplexed) canbe used in conjunction with recent advances in crop genomics. Thesystems described herein can be used to perform efficient and costeffective plant gene or genome interrogation or editing ormanipulation—for instance, for rapid investigation and/or selectionand/or interrogations and/or comparison and/or manipulations and/ortransformation of plant genes or genomes; e.g., to create, identify,develop, optimize, or confer trait(s) or characteristic(s) to plant(s)or to transform a plant genome. There can accordingly be improvedproduction of plants, new plants with new combinations of traits orcharacteristics or new plants with enhanced traits. The Cas13b effectorprotein system(s) can be used with regard to plants in Site-DirectedIntegration (SDI) or Gene Editing (GE) or any Near Reverse Breeding(NRB) or Reverse Breeding (RB) techniques. Aspects of utilizing theherein described Cas13b effector protein systems may be analogous to theuse of the CRISPR-Cas (e.g. CRISPR-Cas9) system in plants, and mentionis made of the University of Arizona website “CRISPR-PLANT”(http://www.genome.arizona.edu/crispr/) (supported by Penn State andAGI). Embodiments of the invention can be used in genome editing inplants or where RNAi or similar genome editing techniques have been usedpreviously; see, e.g., Nekrasov, “Plant genome editing made easy:targeted mutagenesis in model and crop plants using the CRISPR-Cassystem,” Plant Methods 2013, 9:39 (doi:10.1186/1746-4811-9-39); Brooks,“Efficient gene editing in tomato in the first generation using theCRISPR-Cas9 system,” Plant Physiology September 2014 pp 114.247577;Shan, “Targeted genome modification of crop plants using a CRISPR-Cassystem,” Nature Biotechnology 31, 686-688 (2013); Feng, “Efficientgenome editing in plants using a CRISPR/Cas system,” Cell Research(2013) 23:1229-1232. doi:10.1038/cr.2013.114; published online 20 Aug.2013; Xie, “RNA-guided genome editing in plants using a CRISPR-Cassystem,” Mol Plant. 2013 November; 6(6):1975-83. doi: 10.1093/mp/sst119.Epub 2013 Aug. 17; Xu, “Gene targeting using the Agrobacteriumtumefaciens-mediated CRISPR-Cas system in rice,” Rice 2014, 7:5 (2014),Zhou et al., “Exploiting SNPs for biallelic CRISPR mutations in theoutcrossing woody perennial Populus reveals 4-coumarate: CoA ligasespecificity and Redundancy,” New Phytologist (2015) (Forum) 1-4(available online only at www.newphytologist.com); Caliando et al,“Targeted DNA degradation using a CRISPR device stably carried in thehost genome, NATURE COMMUNICATIONS 6:6989, DOI: 10.1038/ncomms7989,www.nature.com/naturecommunications DOI: 10.1038/ncomms7989; U.S. Pat.No. 6,603,061-Agrobacterium-Mediated Plant Transformation Method; U.S.Pat. No. 7,868,149-Plant Genome Sequences and Uses Thereof and US2009/0100536-Transgenic Plants with Enhanced Agronomic Traits, all thecontents and disclosure of each of which are herein incorporated byreference in their entirety. In the practice of the invention, thecontents and disclosure of Morrell et al “Crop genomics: advances andapplications,” Nat Rev Genet. 2011 Dec. 29; 13(2):85-96; each of whichis incorporated by reference herein including as to how hereinembodiments may be used as to plants. Accordingly, reference herein toanimal cells may also apply, mutatis mutandis, to plant cells unlessotherwise apparent; and, the enzymes herein having reduced off-targeteffects and systems employing such enzymes can be used in plantapplications, including those mentioned herein.

Sugano et al. (Plant Cell Physiol. 2014 March; 55(3):475-81. doi:10.1093/pcp/pcu014. Epub 2014 Jan. 18) reports the application ofCRISPR-Cas9 to targeted mutagenesis in the liverwort Marchantiapolymorpha L., which has emerged as a model species for studying landplant evolution. The U6 promoter of M. polymorpha was identified andcloned to express the gRNA. The target sequence of the gRNA was designedto disrupt the gene encoding auxin response factor 1 (ARF1) in M.polymorpha. Using Agrobacterium-mediated transformation, Sugano et al.isolated stable mutants in the gametophyte generation of M. polymorpha.CRISPR-Cas9-based site-directed mutagenesis in vivo was achieved usingeither the Cauliflower mosaic virus 35S or M. polymorpha EFla promoterto express Cas9. Isolated mutant individuals showing an auxin-resistantphenotype were not chimeric. Moreover, stable mutants were produced byasexual reproduction of T1 plants. Multiple arf1 alleles were easilyestablished using CRIPSR/Cas9-based targeted mutagenesis. The Cas13bsystems of the present invention can be used to regulate the same aswell as other genes, and like expression control systems such as RNAiand siRNA, the method of the invention can be inducible and reversible.

Kabadi et al. (Nucleic Acids Res. 2014 Oct. 29; 42(19):e147. doi:10.1093/nar/gku749. Epub 2014 Aug. 13) developed a single lentiviralsystem to express a Cas9 variant, a reporter gene and up to four sgRNAsfrom independent RNA polymerase III promoters that are incorporated intothe vector by a convenient Golden Gate cloning method. Each sgRNA wasefficiently expressed and can mediate multiplex gene editing andsustained transcriptional activation in immortalized and primary humancells. The instant invention can be used to regulate the plant genes ofKabadi.

Xing et al. (BMC Plant Biology 2014, 14:327) developed a CRISPR-Cas9binary vector set based on the pGreen or pCAMBIA backbone, as well as agRNA. This toolkit requires no restriction enzymes besides Bsa1 togenerate final constructs harboring maize-codon optimized Cas9 and oneor more gRNAs with high efficiency in as little as one cloning step. Thetoolkit was validated using maize protoplasts, transgenic maize lines,and transgenic Arabidopsis lines and was shown to exhibit highefficiency and specificity. More importantly, using this toolkit,targeted mutations of three Arabidopsis genes were detected intransgenic seedlings of the T1 generation. Moreover, the multiple-genemutations could be inherited by the next generation. (guide RNA) modulevector set, as a toolkit for multiplex genome editing in plants. TheCas13b systems and proteins of the instant invention may be used totarget the genes targeted by Xing.

The Cas13b CRISPR systems of the invention may be used in the detectionof plant viruses. Gambino et al. (Phytopathology. 2006 November;96(11):1223-9. doi: 10.1094/PHYTO-96-1223) relied on amplification andmultiplex PCR for simultaneous detection of nine grapevine viruses. TheCas13b systems and proteins of the instant invention may similarly beused to detect multiple targets in a host. Moreover, the systems of theinvention can be used to simultaneously knock down viral gene expressionin valuable cultivars, and prevent activation or further infection bytargeting expressed vial RNA.

Murray et al. (Proc Biol Sci. 2013 Jun. 26; 280(1765):20130965. doi:10.1098/rspb.2013.0965; published 2013 Aug. 22) analyzxed 12 plant RNAviruses to investigatge evolutionary rates and found evidence ofepisodic selection possibly due to shifts between different hostgenotyopes or species. The Cas13b systems and proteins of the instantinvention may be used to tarteg or immunize against such viruses in ahost. For example, the systems of the invention can be used to blockviral RNA expression hence replication. Also, the invention can be usedto target nuclic acids for cleavage as wll as to target expression oractivation. Moreover, the systems of the invention can be multiplexed soas to hit multiple targets or multiple isolate of the same virus.

Ma et al. (Mol Plant. 2015 Aug. 3; 8(8):1274-84. doi:10.1016/j.molp.2015.04.007) reports robust CRISPR-Cas9 vector system,utilizing a plant codon optimized Cas9 gene, for convenient andhigh-efficiency multiplex genome editing in monocot and dicot plants. Maet al. designed PCR-based procedures to rapidly generate multiple sgRNAexpression cassettes, which can be assembled into the binary CRISPR-Cas9vectors in one round of cloning by Golden Gate ligation or GibsonAssembly. With this system, Ma et al. edited 46 target sites in ricewith an average 85.4% rate of mutation, mostly in biallelic andhomozygous status. Ma et al. provide examples of loss-of-function genemutations in T0 rice and T1 Arabidopsis plants by simultaneous targetingof multiple (up to eight) members of a gene family, multiple genes in abiosynthetic pathway, or multiple sites in a single gene. Similarly, theCas13b systems of the instant invention can dffieicnelty targetexpression of multiple genes simultaneously.

Lowder et al. (Plant Physiol. 2015 Aug. 21. pii: pp. 00636.2015) alsodeveloped a CRISPR-Cas9 toolbox enables multiplex genome editing andtranscriptional regulation of expressed, silenced or non-coding genes inplants. This toolbox provides researchers with a protocol and reagentsto quickly and efficiently assemble functional CRISPR-Cas9 T-DNAconstructs for monocots and dicots using Golden Gate and Gateway cloningmethods. It comes with a full suite of capabilities, includingmultiplexed gene editing and transcriptional activation or repression ofplant endogenous genes. T-DNA based transformation technology isfundamental to modern plant biotechnology, genetics, molecular biologyand physiology. As such, we developed a method for the assembly of Cas9(WT, nickase or dCas9) and gRNA(s) into a T-DNA destination-vector ofinterest. The assembly method is based on both Golden Gate assembly andMultiSite Gateway recombination. Three modules are required forassembly. The first module is a Cas9 entry vector, which containspromoterless Cas9 or its derivative genes flanked by attL1 and attR5sites. The second module is a gRNA entry vector which contains entrygRNA expression cassettes flanked by attL5 and attL2 sites. The thirdmodule includes attR1-attR2-containing destination T-DNA vectors thatprovide promoters of choice for Cas9 expression. The toolbox of Lowderet al. may be applied to the Cas13b effector protein system of thepresent invention.

Organisms such as yeast and microalgae are widely used for syntheticbiology. Stovicek et al. (Metab. Eng. Comm., 2015; 2:13 describes genomeediting of industrial yeast, for example, Saccharomyces cerevisae, toefficiently produce robust strains for industrial production. Stovicekused a CRISPR-Cas9 system codon-optimized for yeast to simultaneouslydisrupt both alleles of an endogenous gene and knock in a heterologousgene. Cas9 and gRNA were expressed from genomic or episomal 2μ-basedvector locations. The authors also showed that gene disruptionefficiency could be improved by optimization of the levels of Cas9 andgRNA expression. Hlavová et al. (Biotechnol. Adv. 2015) discussesdevelopment of species or strains of microalgae using techniques such asCRISPR to target nuclear and chloroplast genes for insertionalmutagenesis and screening. The same plasmids and vectors can be appliedto the Cas13b systems of the instant invention.

Petersen (“Towards precisely glycol engineered plants,” Plant BiotechDenmark Annual meeting 2015, Copenhagen, Denmark) developed a method ofusing CRISPR/Cas9 to engineer genome changes in Arabidopsis, for exampleto glyco engineer Arabidopsis for production of proteins and productshaving desired posttranslational modifications. Hebelstrup et al. (FrontPlant Sci. 2015 Apr. 23; 6:247) outlines in planta starchbioengineering, providing crops that express starch modifying enzymesand directly produce products that normally are made by industrialchemical and/or physical treatments of starches. The methods of Petersenand Hebelstrup may be applied to the Cas13b effector protein system ofthe present invention.

Kurth et al, J Virol. 2012 June; 86(11):6002-9. doi:10.1128/JVI.00436-12. Epub 2012 Mar. 21) developed an RNA virus-basedvector for the introduction of desired traits into grapevine withoutheritable modifications to the genome. The vector provided the abilityto regulate expression of of endogenous genes by virus-induced genesilencing. The Cas13b systems and proteins of the instant invention canbe used to silence genes and proteins without heritable modification tothe genome.

In an embodiment, the plant may be a legume. The present invention mayutilize the herein disclosed CRISPR-Cas system for exploring andmodifying, for example, without limitation, soybeans, peas, and peanuts.Curtin et al. provides a toolbox for legume function genomics. (SeeCurtin et al., “A genome engineering toolbox for legume Functionalgenomics,” International Plant and Animal Genome Conference XXII 2014).Curtin used the genetic transformation of CRISPR to knock-out/downsingle copy and duplicated legume genes both in hairy root and wholeplant systems. Some of the target genes were chosen in order to exploreand optimize the features of knock-out/down systems (e.g., phytoenedesaturase), while others were identified by soybean homology toArabidopsis Dicer-like genes or by genome-wide association studies ofnodulation in Medicago. The Cas13b systems and proteins of the instantinvention can be used to knockout/knockdown systems.

Peanut allergies and allergies to legumes generally are a real andserious health concern. The Cas13b effector protein system of thepresent invention can be used to identify and then edit or silence genesencoding allergenic proteins of such legumes. Without limitation as tosuch genes and proteins, Nicolaou et al. identifies allergenic proteinsin peanuts, soybeans, lentils, peas, lupin, green beans, and mung beans.See, Nicolaou et al., Current Opinion in Allergy and Clinical Immunology2011; 11(3):222).

In an advantageous embodiment, the plant may be a tree. The presentinvention may also utilize the herein disclosed CRISPR Cas system forherbaceous systems (see, e.g., Belhaj et al., Plant Methods 9: 39 andHarrison et al., Genes & Development 28: 1859-1872). In a particularlyadvantageous embodiment, the CRISPR Cas system of the present inventionmay target single nucleotide polymorphisms (SNPs) in trees (see, e.g.,Zhou et al., New Phytologist, Volume 208, Issue 2, pages 298-301,October 2015). In the Zhou et al. study, the authors applied a CRISPRCas system in the woody perennial Populus using the 4-coumarate:CoAligase (4CL) gene family as a case study and achieved 100% mutationalefficiency for two 4CL genes targeted, with every transformant examinedcarrying biallelic modifications. In the Zhou et al., study, theCRISPR-Cas9 system was highly sensitive to single nucleotidepolymorphisms (SNPs), as cleavage for a third 4CL gene was abolished dueto SNPs in the target sequence. These methods may be applied to theCas13b effector protein system of the present invention.

The methods of Zhou et al. (New Phytologist, Volume 208, Issue 2, pages298-301, October 2015) may be applied to the present invention asfollows. Two 4CL genes, 4CL1 and 4CL2, associated with lignin andflavonoid biosynthesis, respectively are targeted for CRISPR-Cas9editing. The Populus tremula×alba clone 717-1B4 routinely used fortransformation is divergent from the genome-sequenced Populustrichocarpa. Therefore, the 4CL1 and 4CL2 gRNAs designed from thereference genome are interrogated with in-house 717 RNA-Seq data toensure the absence of SNPs which could limit Cas efficiency. A thirdgRNA designed for 4CL5, a genome duplicate of 4CL1, is also included.The corresponding 717 sequence harbors one SNP in each allelenear/within the PAM, both of which are expected to abolish targeting bythe 4CL5-gRNA. All three gRNA target sites are located within the firstexon. For 717 transformation, the gRNA is expressed from the MedicagoU6.6 promoter, along with a human codon-optimized Cas under control ofthe CaMV 35S promoter in a binary vector. Transformation with theCas-only vector can serve as a control. Randomly selected 4CL1 and 4CL2lines are subjected to amplicon-sequencing. The data is then processedand biallelic mutations are confirmed in all cases. These methods may beapplied to the Cas13b effector protein system of the present invention.

In plants, pathogens are often host-specific. For example, Fusariumoxysporum f. sp. lycopersici causes tomato wilt but attacks only tomato,and F. oxysporum f. dianthii Puccinia graminis f sp. tritici attacksonly wheat. Plants have existing and induced defenses to resist mostpathogens. Mutations and recombination events across plant generationslead to genetic variability that gives rise to susceptibility,especially as pathogens reproduce with more frequency than plants. Inplants there can be non-host resistance, e.g., the host and pathogen areincompatible. There can also be Horizontal Resistance, e.g., partialresistance against all races of a pathogen, typically controlled by manygenes and Vertical Resistance, e.g., complete resistance to some racesof a pathogen but not to other races, typically controlled by a fewgenes. In a Gene-for-Gene level, plants and pathogens evolve together,and the genetic changes in one balance changes in other. Accordingly,using Natural Variability, breeders combine most useful genes for Yield,Quality, Uniformity, Hardiness, Resistance. The sources of resistancegenes include native or foreign Varieties, Heirloom Varieties, WildPlant Relatives, and Induced Mutations, e.g., treating plant materialwith mutagenic agents. Using the present invention, plant breeders areprovided with a new tool to induce mutations. Accordingly, one skilledin the art can analyze the genome of sources of resistance genes, and inVarieties having desired characteristics or traits employ the presentinvention to induce the rise of resistance genes, with more precisionthan previous mutagenic agents and hence accelerate and improve plantbreeding programs.

Aside from the plants otherwise discussed herein and above, engineeredplants modified by the effector protein and suitable guide, and progenythereof, as provided. These may include disease or drought resistantcrops, such as wheat, barley, rice, soybean or corn; plants modified toremove or reduce the ability to self-pollinate (but which can instead,optionally, hybridise instead); and allergenic foods such as peanuts andnuts where the immunogenic proteins have been disabled, destroyed ordisrupted by targeting via a effector protein and suitable guide.

Therapeutic Treatment

The system of the invention can be applied in areas of former RNAcutting technologies, without undue experimentation, from thisdisclosure, including therapeutic, assay and other applications, becausethe present application provides the foundation for informed engineeringof the system. The present invention provides for therapeutic treatmentof a disease caused by overexpression of RNA, toxic RNA and/or mutatedRNA (such as, for example, splicing defects or truncations). Expressionof the toxic RNA may be associated with formation of nuclear inclusionsand late-onset degenerative changes in brain, heart or skeletal muscle.In the best studied example, myotonic dystrophy, it appears that themain pathogenic effect of the toxic RNA is to sequester binding proteinsand compromise the regulation of alternative splicing (Hum. Mol. Genet.(2006) 15 (suppl 2): R162-R169). Myotonic dystrophy [dystrophiamyotonica (DM)] is of particular interest to geneticists because itproduces an extremely wide range of clinical features. A partial listingwould include muscle wasting, cataracts, insulin resistance, testicularatrophy, slowing of cardiac conduction, cutaneous tumors and effects oncognition. The classical form of DM, which is now called DM type 1(DM1), is caused by an expansion of CTG repeats in the 3′-untranslatedregion (UTR) of DMPK, a gene encoding a cytosolic protein kinase.

The below table presents a list of exons shown to have misregulatedalternative splicing in DM1 skeletal muscle, heart or brain.

Tissue/gene Target Reference Skeletal muscle ALP ex 5a, 5b Lin X., etal. Failure of MBNL1-dependent postnatal splicing transitions inmyotonic dystrophy. Hum. Mol. Genet 2006; 15: 2087-2097 CAPN3 ex 16 LinX., et al. Failure of MBNL1-dependent postnatal splicing transitions inmyotonic dystrophy. Hum. Mol. Genet 2006; 15: 2087-2097 CLCN1 int 2, ex7a, 8a Mankodi A., et al. Expanded CUG repeats trigger aberrant splicingof ClC-1 chloride channel pre-mRNA and hyperexcitability of skeletalmuscle in myotonic dystrophy. Mol. Cell 2002; 10: 35-44 Charlet-B N., etal. Loss of the muscle-specific chloride channel in type 1 myotonicdystrophy due to misregulated alternative splicing. Mol. Cell 2002; 10:45-53 FHOS ex 11a Lin X., et al. Failure of MBNL1-dependent postnatalsplicing transitions in myotonic dystrophy. Hum. Mol. Genet 2006; 15:2087-2097 GFAT1 ex 10 Lin X., et al. Failure of MBNL1-dependentpostnatal splicing transitions in myotonic dystrophy. Hum. Mol. Genet2006; 15: 2087-2097 IR ex 11 Savkur R. S., et al. Aberrant regulation ofinsulin receptor alternative splicing is associated with insulinresistance in myotonic dystrophy. Nat. Genet. 2001; 29: 40-47 MBNL1 ex 7Lin X., et al. Failure of MBNL1-dependent postnatal splicing transitionsin myotonic dystrophy. Hum. Mol. Genet 2006; 15: 2087-2097 MBNL2 ex 7Lin X., et al. Failure of MBNL1-dependent postnatal splicing transitionsin myotonic dystrophy. Hum. Mol. Genet 2006; 15: 2087-2097 MTMR1 ex 2.1,2.2 Buj-Bello A., et al. Muscle-specific alternative splicing ofmyotubularin-related 1 gene is impaired in DM1 muscle cells. Hum. Mol.Genet. 2002; 11: 2297-2307 NRAP ex 12 Lin X., et al. Failure ofMBNL1-dependent postnatal splicing transitions in myotonic dystrophy.Hum. Mol. Genet 2006; 15: 2087-2097 RYR1 ex 70 Kimura T., et al. AlteredmRNA splicing of the skeletal muscle ryanodine receptor andsarcoplasmic/endoplasmic reticulum Ca2+- ATPase in myotonic dystrophytype 1. Hum. Mol. Genet. 2005; 14: 2189-2200 SERCA1 ex 22 Kimura T., etal. Altered mRNA splicing of the skeletal muscle ryanodine receptor andsarcoplasmic/endoplasmic reticulum Ca2+- ATPase in myotonic dystrophytype 1. Hum. Mol. Genet. 2005; 14: 2189-2200 Lin X., et al. Failure ofMBNL1-dependent postnatal splicing transitions in myotonic dystrophy.Hum. Mol. Genet 2006; 15: 2087-2097 z-Titin ex Zr4, Zr5 Lin X., et al.Failure of MBNL1-dependent postnatal splicing transitions in myotonicdystrophy. Hum. Mol. Genet 2006; 15: 2087-2097 m-Titin M-line ex5 LinX., et al. Failure of MBNL1-dependent postnatal splicing transitions inmyotonic dystrophy. Hum. Mol. Genet 2006; 15: 2087-2097 TNNT3 fetal exKanadia R. N., et al. A muscleblind knockout model for myotonicdystrophy. Science 2003; 302: 1978-1980 ZASP ex 11 Lin X., et al.Failure of MBNL1-dependent postnatal splicing transitions in myotonicdystrophy. Hum. Mol. Genet 2006; 15: 2087-2097 Heart TNNT2 ex 5 PhilipsA. V., et al. Disruption of splicing regulated by a CUG-binding proteinin myotonic dystrophy. Science 1998; 280: 737-741 ZASP ex 11 Mankodi A.,et al. Nuclear RNA foci in the heart in myotonic dystrophy. Circ. Res.2005; 97: 1152-1155 m-Titin M-line ex 5 Mankodi A., et al. Nuclear RNAfoci in the heart in myotonic dystrophy. Circ. Res. 2005; 97: 1152-1155KCNAB1 ex 2 Mankodi A., et al. Nuclear RNA foci in the heart in myotonicdystrophy. Circ. Res. 2005; 97: 1152-1155 ALP ex 5 (Mankodi A., et al.Nuclear RNA foci in the heart in myotonic dystrophy. Circ. Res. 2005;97: 1152-1155 Brain TAU ex 2, ex 10 Sergeant N., et al. Dysregulation ofhuman brain microtubule- associated tau mRNA maturation in myotonicdystrophy type 1. Hum. Mol. Genet. 2001; 10: 2143-2155 Jiang H., et al.Myotonic dystrophy type 1 associated with nuclear foci of mutant RNA,sequestration of muscleblind proteins, and deregulated alternativesplicing in neurons. Hum. Mol. Genet. 2004; 13: 3079-3088 APP ex 7 JiangH., et al. Myotonic dystrophy type 1 associated with nuclear foci ofmutant RNA, sequestration of muscleblind proteins, and deregulatedalternative splicing in neurons. Hum. Mol. Genet. 2004; 13: 3079-3088NMDAR1 ex 5 Jiang H., et al. Myotonic dystrophy type 1 associated withnuclear foci of mutant RNA, sequestration of muscleblind proteins, andderegulated alternative splicing in neurons. Hum. Mol. Genet. 2004; 13:3079-3088

The enzymes of the present invention may target overexpressed RNA ortoxic RNA, such as for example, the DMPK gene or any of the misregulatedalternative splicing in DM1 skeletal muscle, heart or brain in, forexample, the above table.

The enzymes of the present invention may also target trans-actingmutations affecting RNA-dependent functions that cause disease(summarized in Cell. 2009 Feb. 20; 136(4): 777-793) as indicated in thebelow table.

DISEASE GENE/MUTATION FUNCTION Prader Willi syndrome SNORD116 ribosomebiogenesis Spinal muscular atrophy SMN2 splicing (SMA) Dyskeratosiscongenita DKC1 telomerase/ (X-linked) translation Dyskeratosis congenitaTERC telomerase (autosomal dominant) Dyskeratosis congenita TERTtelomerase (autosomal dominant) Diamond-Blackfan anemia RPS19, RPS24ribosome biogenesis Shwachman-Diamond SBDS ribosome biogenesis syndromeTreacher-Collins syndrome TCOF1 ribosome biogenesis Prostate cancerSNHG5 ribosome biogenesis Myotonic dystrophy, type 1 DMPK (RNA gain-of-protein kinase (DM1) function) Myotonic dystrophy type 2 ZNF9 (RNAgain-of- RNA binding (DM2) function) Spinocerebellar ataxia 8ATXN8/ATXN8OS unknown/noncoding (SCA8) (RNA gain-of- RNA function)Huntington's disease-like 2 JPH3 (RNA gain-of- ion channel (HDL2)function) function Fragile X-associated tremor FMR1 (RNA gain-of-translation/mRNA ataxia syndrome (FXTAS) function) localization FragileX syndrome FMR1 translation/mRNA localization X-linked mentalretardation UPF3B translation/nonsense mediated decay Oculopharyngealmuscular PABPN1 3′ end formation dystrophy (OPMD) Human pigmentary DSRADediting genodermatosis Retinitis pigmentosa PRPF31 splicing Retinitispigmentosa PRPF8 splicing Retinitis pigmentosa HPRP3 splicing Retinitispigmentosa PAP1 splicing Cartilage-hair hypoplasia RMRP splicing(recessive) Autism 7q22-q33 locus noncoding RNA breakpointBeckwith-Wiedemann H19 noncoding RNA syndrome (BWS) Charcot-Marie-ToothGRS translation (CMT) Disease Charcot-Marie-Tooth YRS translation (CMT)Disease Amyotrophic lateral TARDBP splicing, sclerosis (ALS)transcription Leukoencephalopathy with EIF2B1 translation vanishingwhite matter Wolcott-Rallison EIF2AK3 translation syndrome (protease)Mitochondrial myopathy PUS1 translation and sideroblastic anemia (MLASA)Encephalomyopathy and TSFM translation hypertrophic (mitochondrial)cardiomyopathy Hereditary spastic SPG7 ribosome biogenesis paraplegiaLeukoencephalopathy DARS2 translation (mitochondrial) Susceptibility toLARS2 translation diabetes mellitus (mitochondrial) Deafness MTRNR1ribosome biogenesis (mitochondrial) MELAS syndrome, MTRNR2 ribosomebiogenesis deafness (mitochondrial) Cancer SFRS1 splicing, translation,export Cancer RBM5 splicing Multiple disorders mitochondrial tRNAtranslation mutations (mitochondrial) Cancer miR-17-92 cluster RNAinterference Cancer miR-372/miR-373 RNA interference

The enzyme of the present invention may also be used in the treatment ofvarious tauopathies, including primary and secondary tauopathies, suchas primary age-related tauopathy (PART)/Neurofibrillarytangle-predominant senile dementia, with NFTs similar to AD, but withoutplaques, dementia pugilistica (chronic traumatic encephalopathy),progressive supranuclear palsy, corticobasal degeneration,frontotemporal dementia and parkinsonism linked to chromosome 17,lytico-Bodig disease (Parkinson-dementia complex of Guam), gangliogliomaand gangliocytoma, meningioangiomatosis, postencephalitic parkinsonism,subacute sclerosing panencephalitis, as well as lead encephalopathy,tuberous sclerosis, Hallervorden-Spatz disease, and lipofuscinosis,alzheimers disease. The enzymes of the present invention may also targetmutations disrupting the cis-acting splicing code cause splicing defectsand disease (summarized in Cell. 2009 Feb. 20; 136(4): 777-793). Themotor neuron degenerative disease SMA results from deletion of the SMN1gene. The remaining SMN2 gene has a C->T substitution in exon 7 thatinactivates an exonic splicing enhancer (ESE), and creates an exonicsplicing silencer (ESS), leading to exon 7 skipping and a truncatedprotein (SMNA7). A T->A substitution in exon 31 of the dystrophin genesimultaneously creates a premature termination codon (STOP) and an ESS,leading to exon 31 skipping. This mutation causes a mild form of DMDbecause the mRNA lacking exon 31 produces a partially functionalprotein. Mutations within and downstream of exon 10 of the MAPT geneencoding the tau protein affect splicing regulatory elements and disruptthe normal 1:1 ratio of mRNAs including or excluding exon 10. Thisresults in a perturbed balance between tau proteins containing eitherfour or three microtubule-binding domains (4R-tau and 3R-tau,respectively), causing the neuropathological disorder FTDP-17. Theexample shown is the N279K mutation which enhances an ESE functionpromoting exon 10 inclusion and shifting the balance toward increased4R-tau. Polymorphic (UG)m(U)n tracts within the 3′ splice site of theCFTR gene exon 9 influence the extent of exon 9 inclusion and the levelof full-length functional protein, modifying the severity of cysticfibrosis (CF) caused by a mutation elsewhere in the CFTR gene.

The innate immune system detects viral infection primarily byrecognizing viral nucleic acids inside an infected cell, referred to asDNA or RNA sensing. In vitro RNA sensing assays can be used to detectspecific RNA substrates. The RNA targeting effector protein can forinstance be used for RNA-based sensing in living cells. Examples ofapplications are diagnostics by sensing of, for examples,disease-specific RNAs.

The RNA targeting effector protein of the invention can further be usedfor antiviral activity, in particular against RNA viruses. The effectorprotein can be targeted to the viral RNA using a suitable guide RNAselective for a selected viral RNA sequence. In particular, the effectorprotein may be an active nuclease that cleaves RNA, such as singlestranded RNA. provided is therefore the use of an RNA targeting effectorprotein of the invention as an antiviral agent.

Therapeutic dosages of the enzyme system of the present invention totarget RNA the above-referenced RNAs are contemplated to be about 0.1 toabout 2 mg/kg the dosages may be administered sequentially with amonitored response, and repeated dosages if necessary, up to about 7 to10 doses per patient. Advantageously, samples are collected from eachpatient during the treatment regimen to ascertain the effectiveness oftreatment. For example, RNA samples may be isolated and quantified todetermine if expression is reduced or ameliorated. Such a diagnostic iswithin the purview of one of skill in the art.

Transcript Detection Methods

The effector proteins and systems of the invention are useful forspecific detection of RNAs in a cell or other sample. In the presence ofan RNA target of interest, guide-dependent Cas13b nuclease activity maybe accompanied by non-specific RNAse activity against collateraltargets. To take advantage of the RNase activity, all that is needed isa reporter substrate that can be detectably cleaved. For example, areporter molecule can comprise RNA, tagged with a fluorescent reportermolecule (fluor) on one end and a quencher on the other. In the absenceof Cas13b RNase activity, the physical proximity of the quencher dampensfluorescence from the fluor to low levels. When Cas13b target specificcleavage is activated by the presence of an RNA target-of-interest andsuitable guide RNA, the RNA-containing reporter molecule isnon-specifically cleaved and the fluor and quencher are spatiallyseparated. This causes the fluor to emit a detectable signal whenexcited by light of the appropriate wavelength.

In an aspect, the invention relates to a (target) RNA detection systemcomprising an RNA targeting effector; one or more guide RNAs designed tobind to the corresponding RNA target; and an RNA-based cleavageinducible reporter construct. In another aspect, the invention relatesto a method for (target) RNA detection in a sample, comprising adding anRNA targeting effector, one or more guide RNAs designed to bind to said(target) RNA, and an RNA-based cleavage inducible reporter construct tosaid sample. In a further aspect, the invention relates to a kit ordevice comprising the (target) RNA detection system as defined herein,or a kit or device comprising at least the RNA targeting effector andthe RNA-based cleavage inducible reporter construct. In a furtheraspect, the invention relates to the use of the RNA targeting system orkit or device as defined herein for (target) RNA detection. The RNAtargeting effector in certain embodiments is an RNA guided RNAse. Incertain embodiments, the RNA targeting effector is is a CRISPR effector.In certain embodiments, the RNA targeting effector is a class 2 CRISPReffector. In certain embodiments, the RNA targeting effector is a class2, type VI-B CRISPR effector. In a preferred embodiment, the RNAtargeting effector is Cas13b. In certain embodiments, the RNA targetingeffector, preferably Cas13b, is derived from a species as describedherein elsewhere. It will be understood that the guide RNA designed tobind to said (target) RNA as described herein is capable of forming acomplex with the RNA targeting effector and wherein the guide RNA insaid complex is capable of binding to a target RNA molecule and wherebythe target RNA is cleaved, as also described herein elsewhere. It willbe understood that the guide RNA typically comprises a guide sequenceand a direct repeat, as described herein elsewhere. In certainembodiments, the one or more guide RNAs are designed to bind to one ormore target molecules that are diagnostic for a disease state. Incertain embodiments, the disease state is infection, such as viral,bacterial, fungal, or parasitic infection. In certain embodiments, thedisease state is characterised by aberrant (target) RNA expression. Incertain embodiments, the disease state is cancer. In certainembodiments, the disease state is autoimmune disease. The RNA-basedcleavage inducible reporter construct comprises RNA and cleavage of theRNA results in a detectable readout, i.e. a detectable signal isgenerated upon cleavage of the RNA. In certain embodiments, theRNA-based cleavage inducible reporter construct comprises a fluorochromeand a quencher. The skilled person will understand that different typesof fluorochromes and corresponding quenchers may be used. The skilledperson will readily envisage other types of inducible reporter systemswhich may be adapted for use in the present RNA cleavage reporterconstructs.

In one exemplary assay method, Cas13b effector,target-of-interest-specific guide RNA, and reporter molecule are addedto a cellular sample. An increase in fluorescence indicates the presenceof the RNA target-of-interest. In another exemplary method, a detectionarray is provided. Each location of the array is provided with Cas13beffector, reporter molecule, and a target-of-interest-specific guideRNA. Depending on the assay to be performed, thetarget-of-interest-specific guide RNAs at each location of the array canbe the same, different, or a combination thereof. Differenttarget-of-interest-specific guide RNAs might be provided, for examplewhen it is desired to test for one or more targets in a single sourcesample. The same target-of-interest-specific guide RNA might be providedat each location, for example when it is desired to test multiplesamples for the same target.

As used herein, a “masking construct” refers to a molecule that can becleaved or otherwise deactivated by an activated CRISPR system effectorprotein described herein. In certain example embodiments, the maskingconstruct is a RNA-based masking construct. The masking constructprevents the generation or detection of a positive detectable signal. Apositive detectable signal may be any signal that can be detected usingoptical, fluorescent, chemiluminescent, electrochemical or otherdetection methods known in the art. The masking construct may preventthe generation of a detectable positive signal or mask the presence of adetectable positive signal until the masking construct is removed orotherwise silenced. The term “positive detectable signal” is used todifferentiate from other detectable signals that may be detectable inthe presence of the masking construct. For example, in certainembodiments a first signal may be detected when the masking agent ispresent (i.e. a negative detectable signal), which then converts to asecond signal (e.g. the positive detectable signal) upon detection ofthe target molecules and cleavage or deactivation of the masking agentby the activated CRISPR effector protein.

In certain example embodiments, the masking construct may suppressgeneration of a gene product. The gene product may be encoded by areporter construct that is added to the sample. The masking constructmay be an interfering RNA involved in a RNA interference pathway, suchas a shRHN or siRNA. The masking construct may also comprise microRNA(miRNA). While present, the masking construct suppresses expression ofthe gene product. The gene product may be a fluorescent protein or otherRNA transcript or proteins that would otherwise be detectable by alabeled probe or antibody but for the presence of the masking construct.Upon activation of the effector protein the masking construct is cleavedor otherwise silenced allowing for expression and detection of the geneproduct as the positive detectable signal.

In certain example embodiments, the masking construct may sequester oneor more reagents needed to generate a detectable positive signal suchthat release of the one or more reagents from the masking constructresults in generation of the detectable positive signal. The one or morereagents may combine to produce a colorimetric signal, achemiluminescent signal, a fluorescent signal, or any other detectablesignal and may comprise any reagents known to be suitable for such apurpose. In certain example embodiments, the one or more reagents aresequestered by RNA aptamers that bind the one or more reagents. The oneor more reagents are released when the effector protein is activatedupon detection of a target molecule. In certain example embodiments, theone or more reagents is a protein, such as an enzyme, capable offacilitating generation of a detectable signal, such as a colorimetric,chemiluminescent, or fluorescent signal, that is inhibited orsequestered such that the protein cannot generate the detectable signalby the binding of one or more RNA aptamers to the protein. Uponactivation of the effector proteins disclosed herein, the RNA aptamersare cleaved or degraded to the extent they no longer inhibit theprotein's ability to generate the detectable signal.

In one embodiment, thrombin is used as a signal amplification enzymewith an inhibitory aptamer, for example having the following sequence:GGGAACAAAGCUGAAGUACUUACCC (SEQ ID NO: 135). When this aptamer iscleaved, thrombin becomes active and will cleave a peptide colorimetricsubstrate (see, e.g.,www.sigmaaldrich.com/catalog/product/sigma/t3068?lang=en&region=US) orfluorescent substrate (see, e.g.,www.sigmaaldrich.com/catalog/product/sigma/b9385?lang=en&region=US). Thecolorimetric substrate, para-nitroanilide (pNA), is covalently linked tothe peptide substrate for thrombin. Upon cleavage by thrombin, pNA isreleased and becomes yellow in color and easily visible by eye. Thefluorescent substrate operates by a similar principle and, upon cleavageby thrombin, releases 7-amino-4-methylcoumarin, a blue fluorophore thatcan be detected using a fluorescence detector. Alternatives to thrombininclude horseradish peroxidase (HRP), β-galactosidase, and calf alkalinephosphatase (CAP) which can similarly be used to generate a colorimetricor fluorescent signal, and be inhibited by an inhibitory aptamer.

In certain example embodiments, the masking construct may be immobilizedon a solid substrate in an individual discrete volume (defined furtherbelow) and sequesters a single reagent. For example, the reagent may bea bead comprising a dye. When sequestered by the immobilized reagent,the individual beads are too diffuse to generate a detectable signal,but upon release from the masking construct are able to generate adetectable signal, for example by aggregation or simple increase insolution concentration. In certain example embodiments, the immobilizedmasking agent is a RNA-based aptamer that can be cleaved by theactivated effector protein upon detection of a target molecule.

In certain other example embodiments, the masking construct binds to animmobilized reagent in solution thereby blocking the ability of thereagent to bind to a separate labeled binding partner that is free insolution. Thus, upon application of a washing step to a sample, thelabeled binding partner can be washed out of the sample in the absenceof a target molecule. However, if the effector protein is activated, themasking construct is cleaved to a degree sufficient to interfere withthe ability of the masking construct to bind the reagent therebyallowing the labeled binding partner to bind to the immobilized reagent.Thus, the labeled binding partner remains after the wash step indicatingthe presence of the target molecule in the sample. In certain aspects,the masking construct that binds the immobilized reagent is a RNAaptamer. The immobilized reagent may be a protein and the labeledminding partner may be a labeled antibody. Alternatively, theimmobilized reagent may be a streptavidin and the labeled bindingpartner may be labeled biotin. The label on the binding partner used inthe above embodiments may be any detectable label known in the art. Inaddition, other known binding partners may be used in accordance withthe overall design described here.

In certain example embodiments, the masking construct may comprise aribozyme. Ribozymes are RNA molecules having catalytic properties. Asribozymes, both naturally and engineered, comprise or consist of RNA,that may be targeted by the effector proteins disclosed herein. Theribozyme may be selected or engineered to catalyze a reaction thateither generates a negative detectable signal or prevents generation ofa positive control signal. Upon deactivation of the ribozyme by theactivated effector protein molecule the reaction generating a negativecontrols signal or preventing generation of a positive detectable signalis removed, thereby allowing a positive detectable signal to bedetected. In one example embodiment, the ribozyme may catalyze acolorimetric reaction causing a solution to appear as a first color.When the ribozyme is deactivated the solution then turns to a secondcolor, the second color being the detectable positive signal. An exampleof how ribozymes can be used to catalyze a colorimetric reaction aredescribed in Zhao et al. “Signal amplification ofglucosamine-6-phosphate based on ribozyme glmS,” Biosens Bioelectron.2014; 16:337-42, and provide an example of how such a system could bemodified to work in the context of the embodiments disclosed herein.Alternatively, ribozymes, when present can generate cleavage productsof, for example, RNA transcripts. Thus, detection of a positivedetectable signal may comprise detection of non-cleaved RNA transcriptsthat are only generated in the absence of the ribozyme.

In one example embodiment, the masking construct comprises a detectionagent that changes color depending on whether the detection agent isaggregated or dispersed in solution. For example, certain nanoparticles,such as colloidal gold, undergo a visible purple to red color shift asthey move from aggregates to dispersed particles. Accordingly, incertain example embodiments, such detection agents may be held inaggregate by one or more bridge molecules. At least a portion of thebridge molecule comprises RNA. Upon activation of the effector proteinsdisclosed herein, the RNA portion of the bridge molecule is cleavedallowing the detection agent to disperse and resulting in thecorresponding change in color. In certain example embodiments the,bridge molecule is a RNA molecule. In certain example embodiments, thedetection agent is a colloidal metal. The colloidal metal material mayinclude water-insoluble metal particles or metallic compounds dispersedin a liquid, a hydrosol, or a metal sol. The colloidal metal may beselected from the metals in groups IA, IB, IIB and IIIB of the periodictable, as well as the transition metals, especially those of group VIII.Preferred metals include gold, silver, aluminum, ruthenium, zinc, iron,nickel and calcium. Other suitable metals also include the following inall of their various oxidation states: lithium, sodium, magnesium,potassium, scandium, titanium, vanadium, chromium, manganese, cobalt,copper, gallium, strontium, niobium, molybdenum, palladium, indium, tin,tungsten, rhenium, platinum, and gadolinium. The metals are preferablyprovided in ionic form, derived from an appropriate metal compound, forexample the Al³⁺, Ru³⁺, Zn²⁺, Fe³⁺, Ni²⁺ and Ca²⁺ ions

In certain other example embodiments, the masking construct may comprisean RNA oligonucleotide to which are attached a detectable label and amasking agent of that detectable label. An example of such a detectablelabel/masking agent pair is a fluorophore and a quencher of thefluorophore. Quenching of the fluorophore can occur as a result of theformation of a non-fluorescent complex between the fluorophore andanother fluorophore or non-fluorescent molecule. This mechanism is knownas ground-state complex formation, static quenching, or contactquenching. Accordingly, the RNA oligonucleotide may be designed so thatthe fluorophore and quencher are in sufficient proximity for contactquenching to occur. Fluorophores and their cognate quenchers are knownin the art and can be selected for this purpose by one having ordinaryskill in the art. The particular fluorophore/quencher pair is notcritical in the context of this invention, only that selection of thefluorophore/quencher pairs ensures masking of the fluorophore. Uponactivation of the effector proteins disclosed herein, the RNAoligonucleotide is cleaved thereby severing the proximity between thefluorophore and quencher needed to maintain the contact quenchingeffect. Accordingly, detection of the fluorophore may be used todetermine the presence of a target molecule in a sample.

In one example embodiment, the masking construct may comprise a quantumdot. The quantum dot may have multiple linker molecules attached to thesurface. At least a portion of the linker molecule comprises RNA. Thelinker molecule is attached to the quantum dot at one end and to one ormore quenchers along the length or at terminal ends of the linker suchthat the quenchers are maintained in sufficient proximity for quenchingof the quantum dot to occur. The linker may be branched. As above, thequantum dot/quencher pair is not critical, only that selection of thequantum dot/quencher pair ensures masking of the fluorophore. Quantumdots and their cognate quenchers are known in the art and can beselected for this purpose by one having ordinary skill in the art. Uponactivation of the effector proteins disclosed herein, the RNA portion ofthe linker molecule is cleaved thereby eliminating the proximity betweenthe quantum dot and one or more quenchers needed to maintain thequenching effect. In one embodiment, the quantum dot is streptavidinconjugated, such as Qdot® 625 Streptavidin Conjugate(www.thermofisher.com/order/catalog/product/A10196). RNA are attachedvia biotin linkers and recruit quenching molecules, with the sequence/5Biosg/UCUCGUACGUUC/3IAbRQSp/ (SEQ ID NO: 136) or/5Biosg/UCUCGUACGUUCUCUCGUACGUUC/3IAbRQSp/ (SEQ ID NO: 137) where/5Biosg/is a biotin tag and/3IAbRQSp/ is an Iowa black quencher. Uponcleavage, the quencher will be released and the quantum dot willfluoresce visibly.

In a similar fashion, fluorescence energy transfer (FRET) may be used togenerate a detectable positive signal. FRET is a non-radiative processby which a photon from an energetically excited fluorophore (i.e. “donorfluorophore”) raises the energy state of an electron in another molecule(i.e. “the acceptor”) to higher vibrational levels of the excitedsinglet state. The donor fluorophore returns to the ground state withoutemitting a fluoresce characteristic of that fluorophore. The acceptorcan be another fluorophore or non-fluorescent molecule. If the acceptoris a fluorophore, the transferred energy is emitted as fluorescencecharacteristic of that fluorophore. If the acceptor is a non-fluorescentmolecule the absorbed energy is loss as heat. Thus, in the context ofthe embodiments disclosed herein, the fluorophore/quencher pair isreplaced with a donor fluorophore/acceptor pair attached to theoligonucleotide molecule. When intact, the masking construct generates afirst signal (negative detectable signal) as detected by thefluorescence or heat emitted from the acceptor. Upon activation of theeffector proteins disclosed herein the RNA oligonucleotide is cleavedand FRET is disrupted such that fluorescence of the donor fluorophore isnow detected (positive detectable signal).

One mode of colorimetric readout for the detection of RNAses is basedupon intercalating dyes, which change their absorbance in response tocleavage of long RNAs to short nucleotides. Several existing dyes withthese properties exist. From Wagner (1983), Pyronine-Y will complex withRNA and form a complex that has an absorbance at 572 nm; cleavage of RNAresults in loss of absorbance and a color change. Greiner-Stoeffele(1996) used methylene blue in a similar fashion, with changes inabsorbance at 688 nm upon RNAse activity.

Another mode of colorimetric readout involves nucleic acid substratesthat change color upon cleavage. Witmer (1991) utilized a syntheticribonucleotide substrate, U-3′-BCIP, that releases a reporter groupafter cleavage, resulting in generation of absorbance at 650 nm.

Deaminase Functionalized CRISPR/Cas13

In certain aspects and embodiments of the invention, the Cas13 proteinas described herein (including for instance Cas13a, Cas13b, or Cas13c,including any orthologue such as those described herein elsewhere),including any Cas13 protein variant (such as functional variants,mutants (including but not limited to catalytically inactive mutants),(functional) domains or truncations (including split Cas13), Cas13fusion proteins (e.g. comprising NLS or NES sequences or any otherfusion proteins described herein elsewhere, etc) as described herein maybe covalently or non-covalently associated or fused to a deaminase or afunctional fragment thereof, such as a catalytically active fragmentthereof. The deaminase may be an adenosine deaminase or a cytidinedeaminase, preferably which deaminase is an RNA specific deaminase. Thedeaminase as described herein may be a truncated or mutated deaminase.It will be understood that whenever reference is made herein toadenosine deaminase, similar considerations apply to cytidine deaminase(and instead of deaminating adenine, cytidine is deaminated).

In certain aspects and embodiments, the invention relates to polynucleicacids encoding such Cas13-deaminase fusion proteins, which mayadvantageously be codon-optimized (or encoding separately Cas13 anddeaminase in case of non-covalent linkage), as well as vectors andvector systems for propagation and/or expression, such as prokaryotic oreukaryotic propagation or expression. Exemplary polynucleic acids, andvectors are described herein elsewhere.

In certain aspects and embodiments, the invention relates to host cells(or progeny thereof), organs, or organisms (or off-spring thereof)comprising the proteins and/or polynucleotides or vectors or vectorsystems described above. Exemplary host cells/organs/organisms, as wellas expression systems are described herein elsewhere.

In certain aspects and embodiments, the invention relates to systems,complexes, or compositions (including kits), such as pharmaceuticalcompositions, comprising such proteins, polynucleic acids, vectors orvector systems, host cells, organs, or organisms. Exemplary systems,complexes, or compositions, such as pharmaceutical compositions aredescribed herein elsewhere. It will be understood that such systems,complexes, or compositions may further include a guide RNA, as describedherein elsewhere, including any variant guide RNA (such as escorted,protected, dead guides, etc., including guided comprising aptamers).

In certain aspects and embodiments, the invention relates to uses of ormethods involving the use of such proteins, polynucleic acids, vectorsor vector systems, host cells, organs, organisms, systems, complexes, orcompositions. Exemplary methods and uses are described herein elsewhere.In particular embodiments, the uses and methods involve modifying anAdenine or cytidine in a target RNA sequence of interest. In particularembodiments, the uses or methods are therapeutic or prophylactic, asalso described herein elsewhere. Advantageously, the uses and methodsmay involve targeted base editing. In one aspect, the inventiondescribed herein provides methods for modifying an adenosine residue ata target locus with the aim of remedying and/or preventing a diseasedcondition that is or is likely to be caused by a G-to-A or C-to-T pointmutation or a pathogenic single nucleotide polymorphism (SNP).Pathogenic G-to-A or C-to-T mutations/SNPs associated with variousdiseases affecting the brain and central nervous system are reported inthe ClinVar database. According to the present invention, any of themutations/SNPs can be targeted.

In general the systems disclosed herein comprise a targeting componentand a base editing component. The targeting component functions tospecifically target the base editing component to a target nucleotidesequence in which one or more nucleotides are to be edited. The baseediting component may then catalyze a chemical reaction to convert afirst nucleotide in the target sequence to a second nucleotide. Forexample, the base editor may catalyze conversion of an adenine such thatit is read as guanine by a cell's transcription or translationmachinery, or vice versa. Likewise, the base editing component maycatalyze conversion of cytidine to a uracil, or vice versa. In certainexample embodiments, the base editor may be derived by starting with aknown base editor, such as an adenine deaminase or cytodine deaminase,and modified using methods such as directed evolution to derive newfunctionalities. Directed evolution techniques are known in the art andmay include those described in WO 2015/184016 “High-Throughput Assemblyof Genetic Permutations.”

In an aspect, the invention relates to a (fusion) protein or proteincomplex, or (a) polynucleotide(s) (including vectors and vector systems)encoding such, comprising (a) a catalytically inactive (dead) Cas13protein; and (b) an (adenosine) deaminase protein or catalytic domainthereof; wherein said (adenosine) deaminase protein or catalytic domainthereof is covalently or non-covalently linked to said dead Cas13protein or is adapted to link thereto after delivery. In certainembodiments, the (fusion) protein or protein complex can bind or isadapted to bind to a guide molecule which comprises a guide sequencelinked to a direct repeat sequence; wherein guide molecule forms acomplex with said dead Cas13 protein and directs said complex to bindsaid target RNA sequence of interest, wherein said guide sequence iscapable of hybridizing with a target sequence comprising said Adenine toform an RNA duplex, wherein said guide sequence comprises a non-pairingCytosine at a position corresponding to said Adenine resulting in an A-Cmismatch in the RNA duplex formed; wherein said (adenosine) deaminaseprotein or catalytic domain thereof deaminates said Adenine in said RNAduplex.

In an aspect, the invention relates to a composition, complex, or systemcomprising (a) a catalytically inactive (dead) Cas13 protein; (b) aguide molecule which comprises a guide sequence linked to a directrepeat sequence; and (c) an (adenosine) deaminase protein or catalyticdomain thereof; wherein said (adenosine) deaminase protein or catalyticdomain thereof is covalently or non-covalently linked to said dead Cas13protein or said guide molecule or is adapted to link thereto afterdelivery; wherein guide molecule forms a complex with said dead Cas13protein and directs said complex to bind said target RNA sequence ofinterest, wherein said guide sequence is capable of hybridizing with atarget sequence comprising said Adenine to form an RNA duplex, whereinsaid guide sequence comprises a non-pairing Cytosine at a positioncorresponding to said Adenine resulting in an A-C mismatch in the RNAduplex formed; wherein said (adenosine) deaminase protein or catalyticdomain thereof deaminates said Adenine in said RNA duplex. The inventionadditionally relates to an engineered, non-naturally occurring (vector)system suitable for modifying an Adenine in a target locus of interest,comprising: a guide molecule which comprises a guide sequence, or anucleotide sequence encoding the guide molecule; a CRISPR-Cas protein,or one or more nucleotide sequences encoding the CRISPR-Cas protein; an(adenosine) deaminase protein or catalytic domain thereof, or one ormore nucleotide sequences encoding; wherein the (adenosine) deaminaseprotein or catalytic domain thereof is covalently or non-covalentlylinked to the CRISPR-Cas protein or the guide molecule or is adapted tolink thereto after delivery; wherein the guide sequence is capable ofhybridizing with a target sequence comprising an Adenine within an RNApolynucleotide of interest, but comprises a Cytosine at the positioncorresponding to the Adenine.

In an aspect, the invention relates to an engineered, non-naturallyoccurring vector system suitable for modifying an Adenine in a targetlocus of interest, comprising one or more vectors comprising: (a) afirst regulatory element operably linked to a nucleotide sequenceencoding said guide molecule which comprises said guide sequence, (b) asecond regulatory element operably linked to a nucleotide sequenceencoding said catalytically inactive Cas13 protein; and (c) a nucleotidesequence encoding an (adenosine) deaminase protein or catalytic domainthereof which is under control of said first or second regulatoryelement or operably linked to a third regulatory element; wherein, ifsaid nucleotide sequence encoding an (adenosine) deaminase protein orcatalytic domain thereof is operably linked to a third regulatoryelement, said (adenosine) deaminase protein or catalytic domain thereofis adapted to link to said guide molecule or said Cas13 protein afterexpression; wherein components (a), (b) and (c) are located on the sameor different vectors of the system.

In an aspect, the invention relates to a method of modifying an Adeninein a target RNA sequence of interest. In particular embodiments, themethod comprises delivering to said target RNA: (a) a catalyticallyinactive (dead) Cas13 protein; (b) a guide molecule which comprises aguide sequence linked to a direct repeat sequence; and (c) an(adenosine) deaminase protein or catalytic domain thereof; wherein said(adenosine) deaminase protein or catalytic domain thereof is covalentlyor non-covalently linked to said dead Cas13 protein or said guidemolecule or is adapted to link thereto after delivery; wherein guidemolecule forms a complex with said dead Cas13 protein and directs saidcomplex to bind said target RNA sequence of interest, wherein said guidesequence is capable of hybridizing with a target sequence comprisingsaid Adenine to form an RNA duplex, wherein said guide sequencecomprises a non-pairing Cytosine at a position corresponding to saidAdenine resulting in an A-C mismatch in the RNA duplex formed; whereinsaid (adenosine) deaminase protein or catalytic domain thereofdeaminates said Adenine in said RNA duplex.

The invention further relates to a method for cell therapy, comprisingadministering to a patient in need thereof the modified cell describedherein, wherein the presence of the modified cell remedies a disease inthe patient. In one embodiment, the modified cell for cell therapy is aCAR-T cell capable of recognizing and/or attacking a tumor cell. Inanother embodiment, the modified cell for cell therapy is a stem cell,such as a neural stem cell, a mesenchymal stem cell, a hematopoieticstem cell, or an iPSC cell.

The invention also relates to a method for knocking-out or knocking-downan undesirable activity of a gene, wherein the deamination of the A atthe transcript of the gene results in a loss of function. For example,in one embodiment, the targeted deamination by thedeaminase-functionalized CRISPR system can cause a nonsense mutationresulting in a premature stop codon in an endogenous gene. This mayalter the expression of the endogenous gene and can lead to a desirabletrait in the edited cell. In another embodiment, the targeteddeamination by the deaminase-functionalized CRISPR system can cause anon-conservative missense mutation resulting in a code for a differentamino acid residue in an endogenous gene. This may alter the function ofthe endogenous gene expressed and can also lead to a desirable trait inthe edited cell.

The deaminase-functionalized CRISPR system described herein can be usedto target a specific Adenine within an RNA polynucleotide sequence fordeamination. For example, the guide molecule can form a complex with theCRISPR-Cas protein and directs the complex to bind a target RNA sequencein the RNA polynucleotide of interest. Because the guide sequence isdesigned to have a non-pairing C, the RNA duplex formed between theguide sequence and the target sequence comprises an A-C mismatch, whichdirects the (adenosine) deaminase to contact and deaminate the Aopposite to the non-pairing C, converting it to a Inosine (I). SinceInosine (I) base pairs with C and functions like G in cellular process,the targeted deamination of A described herein are useful for correctionof undesirable G-A and C-T mutations, as well as for obtaining desirableA-G and T-C mutations.

In certain example embodiment the Cas13 protein is Cas13a, Cas13b or Cas13c.

The (adenosine) deaminase protein or catalytic domain thereof may befused to N- or C-terminus of said dead Cas13 protein. In certain exampleembodiments, the (adenosine) deaminase protein or catalytic domainthereof is fused to said dead Cas13 protein by a linker. The linker maybe (GGGGS)₃₋₁₁ (SEQ ID NOS: 138-143), GSG₅ (SEQ ID NO: 144) orLEPGEKPYKCPECGKSFSQSGALTRHQRTHTR (SEQ ID NO: 145).

In certain example embodiments, the (adenosine) deaminase protein orcatalytic domain thereof is linked to an adaptor protein and said guidemolecule or said dead Cas13 protein comprises an aptamer sequencecapable of binding to said adaptor protein. The adaptor sequence may beselected from MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34,JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5,ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1.

In certain example embodiments, the (adenosine) deaminase protein orcatalytic domain thereof is inserted into an internal loop of said deadCas13 protein. In certain example embodiments, the Cas13a proteincomprises one or more mutations in the two HEPN domains, particularly atposition R474 and R1046 of Cas 13a protein originating from Leptotrichiawadei or amino acid positions corresponding thereto of a Cas13aortholog.

In certain example embodiments, the Cas 13 protein is a Cas13b proteins,and the Cas13b comprises a mutation in one or more of positions R116,H121, R1177, H1182 of Cas13b protein originating from Bergeyellazoohelcum ATCC 43767 or amino acid positions corresponding thereto of aCas13b ortholog. In certain other example embodiments, the mutation isone or more of R116A, H121A, R1177A, H1182A of Cas13b proteinoriginating from Bergeyella zoohelcum ATCC 43767 or amino acid positionscorresponding thereto of a Cas13b ortholog.

In certain example embodiments, the guide sequence has a length of about29-53 nt capable of forming said RNA duplex with said target sequence.In certain other example embodiments, the guide sequence has a length ofabout 40-50 nt capable of forming said RNA duplex with said targetsequence. In certain example embodiments, the distance between saidnon-pairing C and the 5′ end of said guide sequence is 20-30nucleotides.

In certain example embodiments, the (adenosine) deaminase protein orcatalytic domain thereof is a human, cephalopod, or Drosophila(adenosine) deaminase protein or catalytic domain thereof. In certainexample embodiments, the (adenosine) deaminase protein or catalyticdomain thereof has been modified to comprise a mutation at glutamicacid⁴⁸⁸ of the hADAR2-D amino acid sequence, or a corresponding positionin a homologous ADAR protein. In certain example embodiments, theglutamic acid residue may be at position 488 or a corresponding positionin a homologous ADAR protein is replaced by a glutamine residue (E488Q).

In certain other example embodiments, the (adenosine) deaminase proteinor catalytic domain thereof is a mutated hADAR2d comprising mutationE488Q or a mutated hADARld comprising mutation El 008Q.

In certain example embodiments, the guide sequence comprises more thanone mismatch corresponding to different adenosine sites in the targetRNA sequence or wherein two guide molecules are used, each comprising amismatch corresponding to a different adenosine sites in the target RNAsequence.

In certain example embodiments, the Cas13 protein and optionally said(adenosine) deaminase protein or catalytic domain thereof comprise oneor more heterologous nuclear localization signal(s) (NLS(s)).

In certain example embodiments, the method further comprises,determining the target sequence of interest and selecting an (adenosine)deaminase protein or catalytic domain thereof which most efficientlydeaminates said Adenine present in then target sequence.

The target RNA sequence of interest may be within a cell. The cell maybe a eukaryotic cell, a non-human animal cell, a human cell, a plantcell. The target locus of interest may be within an animal or plant.

The target RNA sequence of interest may comprise in an RNApolynucleotide in vitro.

The components of the systems described herein may be delivered to saidcell as a ribonucleoprotein complex or as one or more polynucleotidemolecules, or any other delivery method as described herein elsewhere,including viral or non-viral delivery. The one or more polynucleotidemolecules may comprise one or more mRNA molecules encoding thecomponents. The one or more polynucleotide molecules may be comprisedwithin one or more vectors. The one or more polynucleotide molecules mayfurther comprise one or more regulatory elements operably configured toexpress said Cas13 protein, said guide molecule, and said deaminaseprotein or catalytic domain thereof, optionally wherein said one or moreregulatory elements comprise inducible promoters. The one or morepolynucleotide molecules or said ribonucleoprotein complex may bedelivered via particles, vesicles, or one or more viral vectors. Theparticles may comprise a lipid, a sugar, a metal or a protein. Theparticles may comprise lipid nanoparticles. The vesicles may compriseexosomes or liposomes. The one or more viral vectors may comprise one ormore of adenovirus, one or more lentivirus or one or moreadeno-associated virus.

The methods disclosed herein may be used to modify a cell, a cell lineor an organism by manipulation of one or more target RNA sequences.

In certain example embodiments, the deamination of said Adenine in saidtarget RNA of interest remedies a disease caused by transcriptscontaining a pathogenic G→A or C→T point mutation.

The methods may be be used to treat or prevent a disease, or otherwisealleviate a disease or the severity of a disease, such as in particularby the targeted deamination using the deaminase-functionalized CRISPRsystem, wherein the deamination of the A, which remedies a diseasecaused by transcripts containing a pathogenic G→A or C→T point mutation.In certain example embodiments, the disease is selected fromMeier-Gorlin syndrome, Seckel syndrome 4, Joubert syndrome 5, Lebercongenital amaurosis 10; Charcot-Marie-Tooth disease, type 2;Charcot-Marie-Tooth disease, type 2; Usher syndrome, type 2C;Spinocerebellar ataxia 28; Spinocerebellar ataxia 28; Spinocerebellarataxia 28; Long QT syndrome 2; Sjögren-Larsson syndrome; Hereditaryfructosuria; Hereditary fructosuria; Neuroblastoma; Neuroblastoma;Kallmann syndrome 1; Kallmann syndrome 1; Kallmann syndrome 1;Metachromatic leukodystrophy, Rett syndrome, Amyotrophic lateralsclerosis type 10, Li-Fraumeni syndrome. The disease may be a prematuretermination disease.

The methods disclosed herein, may be used to make a modification thataffects the fertility of an organism. The modification may affectssplicing of said target RNA sequence. The modification may introduce amutation in a transcript introducing an amino acid change and causingexpression of a new antigen in a cancer cell.

In certain example embodiments, the target RNA may be a microRNA orcomprised within a microRNA. In certain example embodiments, thedeamination of said Adenine in said target RNA of interest causes a gainof function or a loss of function of a gene. In certain exampleembodiments, the gene is a gene expressed by a cancer cell.

In another aspect, the invention comprises a modified cell or progenythereof that is obtained using the methods disclosed herein, whereinsaid cell comprises a hypoxanthine or a guanine in replace of saidAdenine in said target RNA of interest compared to a corresponding cellnot subjected to the method. The modified cell or progeny thereof may bea eukaryotic cell an animal cell, a human cell, a therapeutic T cell, anantibody-producing B cell, a plant cell.

In another aspect, the invention comprises a non-human animal comprisingsaid modified cell or progeny thereof. The modified may be a plant cell.

In another aspect, the invention comprises a method for cell therapy,comprising administering to a patient in need thereof the modified cellsdisclosed herein, wherein the presence of said modified cell remedies adisease in the patient.

In another aspect, the invention is directed to an engineered,non-naturally occurring system suitable for modifying an Adenine in atarget locus of interest, comprising A) a guide molecule which comprisesa guide sequence linked to a direct repeat sequence, or a nucleotidesequence encoding said guide molecule; B) a catalytically inactive Cas13protein, or a nucleotide sequence encoding said catalytically inactiveCas13 protein; C) an (adenosine) deaminase protein or catalytic domainthereof, or a nucleotide sequence encoding said (adenosine) deaminaseprotein or catalytic domain thereof; wherein said (adenosine) deaminaseprotein or catalytic domain thereof is covalently or non-covalentlylinked to said Cas13 protein or said guide molecule or is adapted tolink thereto after delivery; wherein said guide sequence is capable ofhybridizing with a target RNA sequence comprising an Adenine to form anRNA duplex, wherein said guide sequence comprises a non-pairing Cytosineat a position corresponding to said Adenine resulting in an A-C mismatchin the RNA duplex formed.

In another aspect, the invention is directed to an engineered,non-naturally occurring vector system suitable for modifying an Adeninein a target locus of interest, comprising the nucleotide sequences ofa), b) and ca

In another aspect, the invention is directed to an engineered,non-naturally occurring vector system, comprising one or more vectorscomprising: a first regulatory element operably linked to a nucleotidesequence encoding said guide molecule which comprises said guidesequence, a second regulatory element operably linked to a nucleotidesequence encoding said catalytically inactive Cas13 protein; and anucleotide sequence encoding an (adenosine) deaminase protein orcatalytic domain thereof which is under control of said first or secondregulatory element or operably linked to a third regulatory element;wherein, if said nucleotide sequence encoding an (adenosine) deaminaseprotein or catalytic domain thereof is operably linked to a thirdregulatory element, said (adenosine) deaminase protein or catalyticdomain thereof is adapted to link to said guide molecule or said Cas13protein after expression; wherein components A), B) and C) are locatedon the same or different vectors of the system.

In another aspect, the invention is directed to in vitro or ex vivo hostcell or progeny thereof or cell line or progeny thereof comprising thesystems disclosed herein. The host cell or progeny thereof may be a aeukaryotice cell, an animal cell, a human cell, or a plant cell.

In one aspect the present invention provides methods for targeteddeamination of adenine in RNA, more particularly in an RNA sequence ofinterest. According to the methods of the invention, the (adenosine)deaminase (AD) protein is recruited specifically to the relevant Adeninein the RNA sequence of interest by a CRISPR-Cas complex which canspecifically bind to a target sequence. In order to achieve this, the(adenosine) deaminase protein can either be covalently linked to theCRISPR-Cas enzyme or be provided as a separate protein, but adapted soas to ensure recruitment thereof to the CRISPR-Cas complex.

In particular embodiments, of the methods of the present invention,recruitment of the (adenosine) deaminase to the target locus is ensuredby fusing the (adenosine) deaminase or catalytic domain thereof to theCRISPR-Cas protein, which is a Cas13 protein. Methods of generating afusion protein from two separate proteins are known in the art andtypically involve the use of spacers or linkers. The Cas13 protein canbe fused to the (adenosine) deaminase protein or catalytic domainthereof on either the N- or C-terminal end thereof. In particularembodiments, the CRISPR-Cas protein is an inactive or dead Cas13 proteinand is linked to the N-terminus of the deaminase protein or itscatalytic domain.

The term “adenosine deaminase” or “adenosine deaminase protein” as usedherein refers to a protein, a polypeptide, or one or more functionaldomain(s) of a protein or a polypeptide that is capable of catalyzing ahydrolytic deamination reaction that converts an adenine (or an adeninemoiety of a molecule) to a hypoxanthine (or a hypoxanthine moiety of amolecule), as shown below. In some embodiments, the adenine-containingmolecule is an adenosine (A), and the hypoxanthine-containing moleculeis an inosine (I). The adenine-containing molecule can bedeoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

According to the present disclosure, adenosine deaminases that can beused in connection with the present disclosure include, but are notlimited to, members of the enzyme family known as adenosine deaminasesthat act on RNA (ADARs), members of the enzyme family known as adenosinedeaminases that act on tRNA (ADATs), and other adenosine deaminasedomain-containing (ADAD) family members. According to the presentdisclosure, the adenosine deaminase is capable of targeting adenine in aRNA/DNA and RNA duplexes. Indeed, Zheng et al. (Nucleic Acids Res. 2017,45(6): 3369-3377) demonstrate that ADARs can carry out adenosine toinosine editing reactions on RNA/DNA and RNA/RNA duplexes. In particularembodiments, the adenosine deaminase has been modified to increase itsability to edit DNA in a RNA/DNAn RNA duplex as detailed herein below.

In some embodiments, the adenosine deaminase is derived from one or moremetazoa species, including but not limited to, mammals, birds, frogs,squids, fish, flies and worms. In some embodiments, the adenosinedeaminase is a human, squid or Drosophila adenosine deaminase.

In some embodiments, the adenosine deaminase is a human ADAR, includinghADAR1, hADAR2, hADAR3. In some embodiments, the adenosine deaminase isa Caenorhabditis elegans ADAR protein, including ADR-1 and ADR-2. Insome embodiments, the adenosine deaminase is a Drosophila ADAR protein,including dAdar. In some embodiments, the adenosine deaminase is a squidLoligo pealeii ADAR protein, including sqADAR2a and sqADAR2b. In someembodiments, the adenosine deaminase is a human ADAT protein. In someembodiments, the adenosine deaminase is a Drosophila ADAT protein. Insome embodiments, the adenosine deaminase is a human ADAD protein,including TENR (hADAD1) and TENRL (hADAD2).

In some embodiments, the adenosine deaminase protein recognizes andconverts one or more target adenosine residue(s) in a double-strandednucleic acid substrate into inosine residues (s). In some embodiments,the double-stranded nucleic acid substrate is a RNA-DNA hybrid duplex.In some embodiments, the adenosine deaminase protein recognizes abinding window on the double-stranded substrate. In some embodiments,the binding window contains at least one target adenosine residue(s). Insome embodiments, the binding window is in the range of about 3 bp toabout 100 bp. In some embodiments, the binding window is in the range ofabout 5 bp to about 50 bp. In some embodiments, the binding window is inthe range of about 10 bp to about 30 bp. In some embodiments, thebinding window is about 1 bp, 2 bp, 3 bp, 5 bp, 7 bp, 10 bp, 15 bp, 20bp, 25 bp, 30 bp, 40 bp, 45 bp, 50 bp, 55 bp, 60 bp, 65 bp, 70 bp, 75bp, 80 bp, 85 bp, 90 bp, 95 bp, or 100 bp.

In some embodiments, the adenosine deaminase protein comprises one ormore deaminase domains. Not intended to be bound by theory, it iscontemplated that the deaminase domain functions to recognize andconvert one or more target adenosine (A) residue(s) contained in adouble-stranded nucleic acid substrate into inosine (I) residues (s). Insome embodiments, the deaminase domain comprises an active center. Insome embodiments, the active center comprises a zinc ion. In someembodiments, during the A-to-I editing process, base pairing at thetarget adenosine residue is disrupted, and the target adenosine residueis “flipped” out of the double helix to become accessible by theadenosine deaminase. In some embodiments, amino acid residues in or nearthe active center interact with one or more nucleotide(s) 5′ to a targetadenosine residue. In some embodiments, amino acid residues in or nearthe active center interact with one or more nucleotide(s) 3′ to a targetadenosine residue. In some embodiments, amino acid residues in or nearthe active center further interact with the nucleotide complementary tothe target adenosine residue on the opposite strand. In someembodiments, the amino acid residues form hydrogen bonds with the 2′hydroxyl group of the nucleotides.

In some embodiments, the adenosine deaminase comprises human ADAR2 fullprotein (hADAR2) or the deaminase domain thereof (hADAR2-D). In someembodiments, the adenosine deaminase is an ADAR family member that ishomologous to hADAR2 or hADAR2-D.

Particularly, in some embodiments, the homologous ADAR protein is humanADAR1 (hADAR1) or the deaminase domain thereof (hADAR1-D). In someembodiments, glycine 1007 of hADAR1-D corresponds to glycine 487hADAR2-D, and glutamic Acid 1008 of hADAR1-D corresponds to glutamicacid 488 of hADAR2-D.

In some embodiments, the adenosine deaminase comprises the wild-typeamino acid sequence of hADAR2-D. In some embodiments, the adenosinedeaminase comprises one or more mutations in the hADAR2-D sequence, suchthat the editing efficiency, and/or substrate editing preference ofhADAR2-D is changed according to specific needs.

Certain mutations of hADAR1 and hADAR2 proteins have been described inKuttan et al., Proc Natl Acad Sci USA. (2012) 109(48):E3295-304; Want etal. ACS Chem Biol. (2015) 10(11):2512-9; and Zheng et al. Nucleic AcidsRes. (2017) 45(6):3369-337, each of which is incorporated herein byreference in its entirety.

In some embodiments, the adenosine deaminase comprises a mutation atglycine336 of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the glycineresidue at position 336 is replaced by an aspartic acid residue (G336D).

In some embodiments, the adenosine deaminase comprises a mutation atGlycine487 of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the glycineresidue at position 487 is replaced by a non-polar amino acid residuewith relatively small side chains. For example, in some embodiments, theglycine residue at position 487 is replaced by an alanine residue(G487A). In some embodiments, the glycine residue at position 487 isreplaced by a valine residue (G487V). In some embodiments, the glycineresidue at position 487 is replaced by an amino acid residue withrelatively large side chains. In some embodiments, the glycine residueat position 487 is replaced by a arginine residue (G487R). In someembodiments, the glycine residue at position 487 is replaced by a lysineresidue (G487K). In some embodiments, the glycine residue at position487 is replaced by a tryptophan residue (G487W). In some embodiments,the glycine residue at position 487 is replaced by a tyrosine residue(G487Y).

In some embodiments, the adenosine deaminase comprises a mutation atglutamic acid 488 of the hADAR2-D amino acid sequence, or acorresponding position in a homologous ADAR protein. In someembodiments, the glutamic acid residue at position 488 is replaced by aglutamine residue (E488Q). In some embodiments, the glutamic acidresidue at position 488 is replaced by a histidine residue (E488H). Insome embodiments, the glutamic acid residue at position 488 is replaceby an arginine residue (E488R). In some embodiments, the glutamic acidresidue at position 488 is replace by a lysine residue (E488K). In someembodiments, the glutamic acid residue at position 488 is replace by anasparagine residue (E488N). In some embodiments, the glutamic acidresidue at position 488 is replace by an alanine residue (E488A). Insome embodiments, the glutamic acid residue at position 488 is replaceby a Methionine residue (E488M). In some embodiments, the glutamic acidresidue at position 488 is replace by a serine residue (E488S). In someembodiments, the glutamic acid residue at position 488 is replace by aphenylalanine residue (E488F). In some embodiments, the glutamic acidresidue at position 488 is replace by a lysine residue (E488L). In someembodiments, the glutamic acid residue at position 488 is replace by atryptophan residue (E488W).

In some embodiments, the adenosine deaminase comprises a mutation atthreonine 490 of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, thethreonine residue at position 490 is replaced by a cysteine residue(T490C). In some embodiments, the threonine residue at position 490 isreplaced by a serine residue (T490S). In some embodiments, the threonineresidue at position 490 is replaced by an alanine residue (T490A). Insome embodiments, the threonine residue at position 490 is replaced by aphenylalanine residue (T490F). In some embodiments, the threonineresidue at position 490 is replaced by a tyrosine residue (T490Y). Insome embodiments, the threonine residue at position 490 is replaced by aserine residue (T490R). In some embodiments, the threonine residue atposition 490 is replaced by an alanine residue (T490K). In someembodiments, the threonine residue at position 490 is replaced by aphenylalanine residue (T490P). In some embodiments, the threonineresidue at position 490 is replaced by a tyrosine residue (T490E).

In some embodiments, the adenosine deaminase comprises a mutation atvaline493 of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the valineresidue at position 493 is replaced by an alanine residue (V493A). Insome embodiments, the valine residue at position 493 is replaced by aserine residue (V493S). In some embodiments, the valine residue atposition 493 is replaced by a threonine residue (V493T). In someembodiments, the valine residue at position 493 is replaced by anarginine residue (V493R). In some embodiments, the valine residue atposition 493 is replaced by an aspartic acid residue (V493D). In someembodiments, the valine residue at position 493 is replaced by a prolineresidue (V493P). In some embodiments, the valine residue at position 493is replaced by a glycine residue (V493G).

In some embodiments, the adenosine deaminase comprises a mutation atalanine 589 of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the alanineresidue at position 589 is replaced by a valine residue (A589V).

In some embodiments, the adenosine deaminase comprises a mutation atasparagine 597 of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, theasparagine residue at position 597 is replaced by a lysine residue(N597K). In some embodiments, the adenosine deaminase comprises amutation at position 597 of the amino acid sequence, which has anasparagine residue in the wild type sequence. In some embodiments, theasparagine residue at position 597 is replaced by an arginine residue(N597R). In some embodiments, the adenosine deaminase comprises amutation at position 597 of the amino acid sequence, which has anasparagine residue in the wild type sequence. In some embodiments, theasparagine residue at position 597 is replaced by an alanine residue(N597A). In some embodiments, the adenosine deaminase comprises amutation at position 597 of the amino acid sequence, which has anasparagine residue in the wild type sequence. In some embodiments, theasparagine residue at position 597 is replaced by a glutamic acidresidue (N597E). In some embodiments, the adenosine deaminase comprisesa mutation at position 597 of the amino acid sequence, which has anasparagine residue in the wild type sequence. In some embodiments, theasparagine residue at position 597 is replaced by a histidine residue(N597H). In some embodiments, the adenosine deaminase comprises amutation at position 597 of the amino acid sequence, which has anasparagine residue in the wild type sequence. In some embodiments, theasparagine residue at position 597 is replaced by a glycine residue(N597G). In some embodiments, the adenosine deaminase comprises amutation at position 597 of the amino acid sequence, which has anasparagine residue in the wild type sequence. In some embodiments, theasparagine residue at position 597 is replaced by a tyrosine residue(N597Y). In some embodiments, the asparagine residue at position 597 isreplaced by a phenylalanine residue (N597F).

In some embodiments, the adenosine deaminase comprises a mutation atserine599 of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the serineresidue at position 599 is replaced by a threonine residue (S599T).

In some embodiments, the adenosine deaminase comprises a mutation atasparagine613 of the hADAR2-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, theasparagine residue at position 613 is replaced by a lysine residue(N613K). In some embodiments, the adenosine deaminase comprises amutation at position 613 of the amino acid sequence, which has anasparagine residue in the wild type sequence. In some embodiments, theasparagine residue at position 613 is replaced by an arginine residue(N613R). In some embodiments, the adenosine deaminase comprises amutation at position 613 of the amino acid sequence, which has anasparagine residue in the wild type sequence. In some embodiments, theasparagine residue at position 613 is replaced by an alanine residue(N613A) In some embodiments, the adenosine deaminase comprises amutation at position 613 of the amino acid sequence, which has anasparagine residue in the wild type sequence. In some embodiments, theasparagine residue at position 613 is replaced by a glutamic acidresidue (N613E).

In some embodiments, to improve editing efficiency, the adenosinedeaminase may comprise one or more of the mutations: G336D, G487A,G487V, E488Q, E488H, E488R, E488N, E488A, E488S, E488M, T490C, T490S,V493T, V493S, V493A, V493R, V493D, V493P, V493G, N597K, N597R, N597A,N597E, N597H, N597G, N597Y, A589V, S599T, N613K, N613R, N613A, N613E,based on amino acid sequence positions of hADAR2-D, and mutations in ahomologous ADAR protein corresponding to the above.

In some embodiments, to reduce editing efficiency, the adenosinedeaminase may comprise one or more of the mutations: E488F, E488L,E488W, T490A, T490F, T490Y, T490R, T490K, T490P, T490E, N597F, based onamino acid sequence positions of hADAR2-D, and mutations in a homologousADAR protein corresponding to the above. In particular embodiments, itcan be of interest to use an adenosine deaminase enzyme with reducedefficacy to reduce off-target effects.

In certain embodiments, improvement of editing and reduction ofoff-target modification is achieved by chemical modification of gRNAs.gRNAs which are chemically modified as exemplified in Vogel et al.(2014), Angew Chem Int Ed, 53:6267-6271, doi:10.1002/anie.201402634(incorporated herein by reference in its entirety) reduce off-targetactivity and improve on-target efficiency. 2′-O-methyl andphosphothioate modified guide RNAs in general improve editing efficiencyin cells.

ADAR has been known to demonstrate a preference for neighboringnucleotides on either side of the edited A(www.nature.com/nsmb/journal/v23/n5/full/nsmb.3203.html, Matthews et al.(2017), Nature Structural Mol Biol, 23(5): 426-433, incorporated hereinby reference in its entirety). Accordingly, in certain embodiments, thegRNA, target, and/or ADAR is selected optimized for motif preference.

Intentional mismatches have been demonstrated in vitro to allow forediting of non-preferred motifs(https://academic.oup.com/nar/article-lookup/doi/10.1093/nar/gku272;Schneider et al (2014), Nucleic Acid Res, 42(10):e87); Fukuda et al.(2017), Scientific Reports, 7, doi:10.1038/srep41478, incorporatedherein by reference in its entirety). Accordingly, in certainembodiments, to enhance RNA editing efficiency on non-preferred 5′ or 3′neighboring bases, intentional mismatches in neighboring bases areintroduced.

Results suggest that As opposite Cs in the targeting window of the ADARdeaminase domain are preferentially edited over other bases.Additionally, As base-paired with Us within a few bases of the targetedbase show low levels of editing by Cas13b-ADAR fusions, suggesting thatthere is flexibility for the enzyme to edit multiple A's. See e.g. FIG.18. These two observations suggest that multiple As in the activitywindow of Cas13b-ADAR fusions could be specified for editing bymismatching all As to be edited with Cs. Accordingly, in certainembodiments, multiple A:C mismatches in the activity window are designedto create multiple A:I edits. In certain embodiments, to suppresspotential off-target editing in the activity window, non-target As arepaired with As or Gs.

The terms “editing specificity” and “editing preference” are usedinterchangeably herein to refer to the extent of A-to-I editing at aparticular adenosine site in a double-stranded substrate. In someembodiment, the substrate editing preference is determined by the 5′nearest neighbor and/or the 3′ nearest neighbor of the target adenosineresidue. In some embodiments, the adenosine deaminase has preference forthe 5′ nearest neighbor of the substrate ranked as U>A>C>G (“>”indicates greater preference). In some embodiments, the adenosinedeaminase has preference for the 3′ nearest neighbor of the substrateranked as G>C˜A>U (“>” indicates greater preference; “˜” indicatessimilar preference). In some embodiments, the adenosine deaminase haspreference for the 3′ nearest neighbor of the substrate ranked asG>C>U˜A (“>” indicates greater preference; “˜” indicates similarpreference). In some embodiments, the adenosine deaminase has preferencefor the 3′ nearest neighbor of the substrate ranked as G>C>A>U (“>”indicates greater preference). In some embodiments, the adenosinedeaminase has preference for the 3′ nearest neighbor of the substrateranked as C˜G˜A>U (“>” indicates greater preference; “˜” indicatessimilar preference). In some embodiments, the adenosine deaminase haspreference for a triplet sequence containing the target adenosineresidue ranked as TAG>AAG>CAC>AAT>GAA>GAC (“>” indicates greaterpreference), the center A being the target adenosine residue.

In some embodiments, the substrate editing preference of an adenosinedeaminase is affected by the presence or absence of a nucleic acidbinding domain in the adenosine deaminase protein. In some embodiments,to modify substrate editing preference, the deaminase domain isconnected with a double-strand RNA binding domain (dsRBD) or adouble-strand RNA binding motif (dsRBM). In some embodiments, the dsRBDor dsRBM may be derived from an ADAR protein, such as hADAR1 or hADAR2.In some embodiments, a full length ADAR protein that comprises at leastone dsRBD and a deaminase domain is used. In some embodiments, the oneor more dsRBM or dsRBD is at the N-terminus of the deaminase domain. Inother embodiments, the one or more dsRBM or dsRBD is at the C-terminusof the deaminase domain.

In some embodiments, the substrate editing preference of an adenosinedeaminase is affected by amino acid residues near or in the activecenter of the enzyme. In some embodiments, to modify substrate editingpreference, the adenosine deaminase may comprise one or more of themutations: G336D, G487R, G487K, G487W, G487Y, E488Q, E488N, T490A,V493A, V493T, V493S, N597K, N597R, A589V, S599T, N613K, N613R, based onamino acid sequence positions of hADAR2-D, and mutations in a homologousADAR protein corresponding to the above.

Particularly, in some embodiments, to reduce editing specificity, theadenosine deaminase can comprise one or more of mutations E488Q, V493A,N597K, N613K, based on amino acid sequence positions of hADAR2-D, andmutations in a homologous ADAR protein corresponding to the above. Insome embodiments, to increase editing specificity, the adenosinedeaminase can comprise mutation T490A.

In some embodiments, to reduce off-target effects, the adenosinedeaminase comprises one or more of mutations at R348, V351, T375, K376,E396, C451, R455, N473, R474, K475, R477, R481, 5486, E488, T490, 5495,R510, based on amino acid sequence positions of hADAR2-D, and mutationsin a homologous ADAR protein corresponding to the above. In someembodiments, the adenosine deaminase comprises mutation at E488 and oneor more additional positions selected from R348, V351, T375, K376, E396,C451, R455, N473, R474, K475, R477, R481, S486, T490, S495, R510. Insome embodiments, the adenosine deaminase comprises mutation at T375,and optionally at one or more additional positions. In some embodiments,the adenosine deaminase comprises mutation at N473, and optionally atone or more additional positions. In some embodiments, the adenosinedeaminase comprises mutation at V351, and optionally at one or moreadditional positions. In some embodiments, the adenosine deaminasecomprises mutation at E488 and T375, and optionally at one or moreadditional positions. In some embodiments, the adenosine deaminasecomprises mutation at E488 and N473, and optionally at one or moreadditional positions. In some embodiments, the adenosine deaminasecomprises mutation E488 and V351, and optionally at one or moreadditional positions. In some embodiments, the adenosine deaminasecomprises mutation at E488 and one or more of T375, N473, and V351.

In some embodiments, to reduce off-target effects, the adenosinedeaminase comprises one or more of mutations selected from R348E, V351L,T375G, T375S, R455G, R455S, R455E, N473D, R474E, K475Q, R477E, R481E,S486T, E488Q, T490A, T490S, S495T, and R510E, based on amino acidsequence positions of hADAR2-D, and mutations in a homologous ADARprotein corresponding to the above. In some embodiments, the adenosinedeaminase comprises mutation E488Q and one or more additional mutationsselected from R348E, V351L, T375G, T375S, R455G, R455S, R455E, N473D,R474E, K475Q, R477E, R481E, S486T, T490A, T490S, S495T, and R510E. Insome embodiments, the adenosine deaminase comprises mutation T375G orT375S, and optionally one or more additional mutations. In someembodiments, the adenosine deaminase comprises mutation N473D, andoptionally one or more additional mutations. In some embodiments, theadenosine deaminase comprises mutation V351L, and optionally one or moreadditional mutations. In some embodiments, the adenosine deaminasecomprises mutation E488Q, and T375G or T375G, and optionally one or moreadditional mutations. In some embodiments, the adenosine deaminasecomprises mutation E488Q and N473D, and optionally one or moreadditional mutations. In some embodiments, the adenosine deaminasecomprises mutation E488Q and V351L, and optionally one or moreadditional mutations. In some embodiments, the adenosine deaminasecomprises mutation E488Q and one or more of T375G/S, N473D and V351L.

In some embodiments, to increase editing preference for target adenosine(A) with an immediate 5′ G, such as substrates comprising the tripletsequence GAC, the center A being the target adenosine residue, theadenosine deaminase can comprise one or more of mutations G336D, E488Q,E488N, V493T, V493S, V493A, A589V, N597K, N597R, S599T, N613K, N613R,based on amino acid sequence positions of hADAR2-D, and mutations in ahomologous ADAR protein corresponding to the above.

Particularly, in some embodiments, the adenosine deaminase comprisesmutation E488Q or a corresponding mutation in a homologous ADAR proteinfor editing substrates comprising the following triplet sequences: GAC,GAA, GAU, GAG, CAU, AAU, UAC, the center A being the target adenosineresidue.

In some embodiments, the adenosine deaminase comprises the wild-typeamino acid sequence of hADAR1-D. In some embodiments, the adenosinedeaminase comprises one or more mutations in the hADAR1-D sequence, suchthat the editing efficiency, and/or substrate editing preference ofhADAR1-D is changed according to specific needs.

In some embodiments, the adenosine deaminase comprises a mutation atGlycine1007 of the hADAR1-D amino acid sequence, or a correspondingposition in a homologous ADAR protein. In some embodiments, the glycineresidue at position 1007 is replaced by a non-polar amino acid residuewith relatively small side chains. For example, in some embodiments, theglycine residue at position 1007 is replaced by an alanine residue(G1007A). In some embodiments, the glycine residue at position 1007 isreplaced by a valine residue (G1007V). In some embodiments, the glycineresidue at position 1007 is replaced by an amino acid residue withrelatively large side chains. In some embodiments, the glycine residueat position 1007 is replaced by an arginine residue (G1007R). In someembodiments, the glycine residue at position 1007 is replaced by alysine residue (G1007K). In some embodiments, the glycine residue atposition 1007 is replaced by a tryptophan residue (G1007W). In someembodiments, the glycine residue at position 1007 is replaced by atyrosine residue (G1007Y). Additionally, in other embodiments, theglycine residue at position 1007 is replaced by a leucine residue(G1007L). In other embodiments, the glycine residue at position 1007 isreplaced by a threonine residue (G1007T). In other embodiments, theglycine residue at position 1007 is replaced by a serine residue(G1007S).

In some embodiments, the adenosine deaminase comprises a mutation atglutamic acid 1008 of the hADAR1-D amino acid sequence, or acorresponding position in a homologous ADAR protein. In someembodiments, the glutamic acid residue at position 1008 is replaced by apolar amino acid residue having a relatively large side chain. In someembodiments, the glutamic acid residue at position 1008 is replaced by aglutamine residue (E1008Q). In some embodiments, the glutamic acidresidue at position 1008 is replaced by a histidine residue (E1008H). Insome embodiments, the glutamic acid residue at position 1008 is replacedby an arginine residue (E1008R). In some embodiments, the glutamic acidresidue at position 1008 is replaced by a lysine residue (E1008K). Insome embodiments, the glutamic acid residue at position 1008 is replacedby a nonpolar or small polar amino acid residue. In some embodiments,the glutamic acid residue at position 1008 is replaced by aphenylalanine residue (E1008F). In some embodiments, the glutamic acidresidue at position 1008 is replaced by a tryptophan residue (E1008W).In some embodiments, the glutamic acid residue at position 1008 isreplaced by a glycine residue (E1008G). In some embodiments, theglutamic acid residue at position 1008 is replaced by an isoleucineresidue (E10081). In some embodiments, the glutamic acid residue atposition 1008 is replaced by a valine residue (E1008V). In someembodiments, the glutamic acid residue at position 1008 is replaced by aproline residue (E1008P). In some embodiments, the glutamic acid residueat position 1008 is replaced by a serine residue (E1008S). In otherembodiments, the glutamic acid residue at position 1008 is replaced byan asparagine residue (E1008N). In other embodiments, the glutamic acidresidue at position 1008 is replaced by an alanine residue (E1008A). Inother embodiments, the glutamic acid residue at position 1008 isreplaced by a Methionine residue (E1008M). In some embodiments, theglutamic acid residue at position 1008 is replaced by a leucine residue(E1008L).

In some embodiments, to improve editing efficiency, the adenosinedeaminase may comprise one or more of the mutations: E1007S, E1007A,E1007V, E1008Q, E1008R, E1008H, E1008M, E1008N, E1008K, based on aminoacid sequence positions of hADAR1-D, and mutations in a homologous ADARprotein corresponding to the above.

In some embodiments, to reduce editing efficiency, the adenosinedeaminase may comprise one or more of the mutations: E1007R, E1007K,E1007Y, E1007L, E1007T, E1008G, E10081, E1008P, E1008V, E1008F, E1008W,E1008S, E1008N, E1008K, based on amino acid sequence positions ofhADAR1-D, and mutations in a homologous ADAR protein corresponding tothe above.

In some embodiments, the substrate editing preference, efficiency and/orselectivity of an adenosine deaminase is affected by amino acid residuesnear or in the active center of the enzyme. In some embodiments, theadenosine deaminase comprises a mutation at the glutamic acid 1008position in hADAR1-D sequence, or a corresponding position in ahomologous ADAR protein. In some embodiments, the mutation is E1008R, ora corresponding mutation in a homologous ADAR protein. In someembodiments, the E1008R mutant has an increased editing efficiency fortarget adenosine residue that has a mismatched G residue on the oppositestrand.

In some embodiments, the adenosine deaminase protein further comprisesor is connected to one or more double-stranded RNA (dsRNA) bindingmotifs (dsRBMs) or domains (dsRBDs) for recognizing and binding todouble-stranded nucleic acid substrates. In some embodiments, theinteraction between the adenosine deaminase and the double-strandedsubstrate is mediated by one or more additional protein factor(s),including a CRISPR/CAS protein factor. In some embodiments, theinteraction between the adenosine deaminase and the double-strandedsubstrate is further mediated by one or more nucleic acid component(s),including a guide RNA.

In certain example embodiments, directed evolution may be used to designmodified ADAR proteins capable of catalyzing additional reactionsbesides deamination of a adenine to a hypoxanthine. For example

According to the present invention, the substrate of the adenosinedeaminase is an RNA/DNAn RNA duplex formed upon binding of the guidemolecule to its DNA target which then forms the CRISPR-Cas complex withthe CRISPR-Cas enzyme. The RNA/DNA or DNA/RNAn RNA duplex is alsoreferred to herein as the “RNA/DNA hybrid”, “DNA/RNA hybrid” or“double-stranded substrate”. The particular features of the guidemolecule and CRISPR-Cas enzyme are detailed below.

The term “editing selectivity” as used herein refers to the fraction ofall sites on a double-stranded substrate that is edited by an adenosinedeaminase. Without being bound by theory, it is contemplated thatediting selectivity of an adenosine deaminase is affected by thedouble-stranded substrate's length and secondary structures, such as thepresence of mismatched bases, bulges and/or internal loops.

In some embodiments, when the substrate is a perfectly base-pairedduplex longer than 50 bp, the adenosine deaminase may be able todeaminate multiple adenosine residues within the duplex (e.g., 50% ofall adenosine residues). In some embodiments, when the substrate isshorter than 50 bp, the editing selectivity of an adenosine deaminase isaffected by the presence of a mismatch at the target adenosine site.Particularly, in some embodiments, adenosine (A) residue having amismatched cytidine (C) residue on the opposite strand is deaminatedwith high efficiency. In some embodiments, adenosine (A) residue havinga mismatched guanosine (G) residue on the opposite strand is skippedwithout editing.

With respect to general information on CRISPR-Cas Systems, componentsthereof, and delivery of such components, including methods, materials,delivery vehicles, vectors, particles, AAV, and making and usingthereof, including as to amounts and formulations, all useful in thepractice of the instant invention, reference is made to: U.S. Pat. Nos.8,999,641, 8,993,233, 8,945,839, 8,932,814, 8,906,616, 8,895,308,8,889,418, 8,889,356, 8,871,445, 8,865,406, 8,795,965, 8,771,945 and8,697,359; US Patent Publications US 2014-0310830 (U.S. application Ser.No. 14/105,031), US 2014-0287938 A1 (U.S. application Ser. No.14/213,991), US 2014-0273234 A1 (U.S. application Ser. No. 14/293,674),US2014-0273232 A1 (U.S. application Ser. No. 14/290,575), US2014-0273231 (U.S. application Ser. No. 14/259,420), US 2014-0256046 A1(U.S. application Ser. No. 14/226,274), US 2014-0248702 A1 (U.S.application Ser. No. 14/258,458), US 2014-0242700 A1 (U.S. applicationSer. 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Nos. 61/915,150, 61/915,301,61/915,267 and 61/915,260, each filed Dec. 12, 2013; 61/757,972 and61/768,959, filed on Jan. 29, 2013 and Feb. 25, 2013; 61/835,936,61/836,127, 61/836,101, 61/836,080, 61/835,973, and 61/835,931, filedJun. 17, 2013; 62/010,888 and 62/010,879, both filed Jun. 11, 2014;62/010,329 and 62/010,441, each filed Jun. 10, 2014; 61/939,228 and61/939,242, each filed Feb. 12, 2014; 61/980,012, filed Apr. 15, 2014;62/038,358, filed Aug. 17, 2014; 62/054,490, 62/055,484, 62/055,460 and62/055,487, each filed Sep. 25, 2014; and 62/069,243, filed Oct. 27,2014. Reference is also made to U.S. provisional patent applicationsNos. 62/055,484, 62/055,460, and 62/055,487, filed Sep. 25, 2014; U.S.provisional patent application 61/980,012, filed Apr. 15, 2014; and U.S.provisional patent application 61/939,242 filed Feb. 12, 2014. Referenceis made to PCT application designating, inter alia, the United States,application No. PCT/US14/41806, filed Jun. 10, 2014. Reference is madeto U.S. provisional patent application 61/930,214 filed on Jan. 22,2014. Reference is made to U.S. provisional patent applications61/915,251; 61/915,260 and 61/915,267, each filed on Dec. 12, 2013.Reference is made to US provisional patent application U.S. Ser. No.61/980,012 filed Apr. 15, 2014. Reference is made to PCT applicationdesignating, inter alia, the United States, application No.PCT/US14/41806, filed Jun. 10, 2014. Reference is made to U.S.provisional patent application 61/930,214 filed on Jan. 22, 2014.Reference is made to U.S. provisional patent applications 61/915,251;61/915,260 and 61/915,267, each filed on Dec. 12, 2013.

Mention is also made of U.S. application 62/091,455, filed, 12 Dec.2014, PROTECTED GUIDE RNAS (PGRNAS); U.S. application 62/096,708, 24Dec. 2014, PROTECTED GUIDE RNAS (PGRNAS); U.S. application 62/091,462,12 Dec. 2014, DEAD GUIDES FOR CRISPR TRANSCRIPTION FACTORS; U.S.application 62/096,324, 23 Dec. 2014, DEAD GUIDES FOR CRISPRTRANSCRIPTION FACTORS; U.S. application 62/091,456, 12 Dec. 2014,ESCORTED AND FUNCTIONALIZED GUIDES FOR CRISPR-CAS SYSTEMS; U.S.application 62/091,461, 12 Dec. 2014, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR GENOMEEDITING AS TO HEMATOPOETIC STEM CELLS (HSCs); U.S. application62/094,903, 19 Dec. 2014, UNBIASED IDENTIFICATION OF DOUBLE-STRANDBREAKS AND GENOMIC REARRANGEMENT BY GENOME-WISE INSERT CAPTURESEQUENCING; U.S. application 62/096,761, 24 Dec. 2014, ENGINEERING OFSYSTEMS, METHODS AND OPTIMIZED ENZYME AND GUIDE SCAFFOLDS FOR SEQUENCEMANIPULATION; U.S. application 62/098,059, 30 Dec. 2014, RNA-TARGETINGSYSTEM; U.S. application 62/096,656, 24 Dec. 2014, CRISPR HAVING ORASSOCIATED WITH DESTABILIZATION DOMAINS; U.S. application 62/096,697, 24Dec. 2014, CRISPR HAVING OR ASSOCIATED WITH AAV; U.S. application62/098,158, 30 Dec. 2014, ENGINEERED CRISPR COMPLEX INSERTIONALTARGETING SYSTEMS; U.S. application 62/151,052, 22 Apr. 2015, CELLULARTARGETING FOR EXTRACELLULAR EXOSOMAL REPORTING; U.S. application62/054,490, 24 Sep. 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OFTHE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETING DISORDERS ANDDISEASES USING PARTICLE DELIVERY COMPONENTS; U.S. application62/055,484, 25 Sep. 2014, SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCEMANIPULATION WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S.application 62/087,537, 4 Dec. 2014, SYSTEMS, METHODS AND COMPOSITIONSFOR SEQUENCE MANIPULATION WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS;U.S. application 62/054,651, 24 Sep. 2014, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR MODELINGCOMPETITION OF MULTIPLE CANCER MUTATIONS IN VIVO; U.S. application62/067,886, 23 Oct. 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OFTHE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR MODELING COMPETITION OFMULTIPLE CANCER MUTATIONS IN VIVO; U.S. application 62/054,675, 24 Sep.2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CASSYSTEMS AND COMPOSITIONS IN NEURONAL CELLS/TISSUES; U.S. application62/054,528, 24 Sep. 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OFTHE CRISPR-CAS SYSTEMS AND COMPOSITIONS IN IMMUNE DISEASES OR DISORDERS;U.S. application 62/055,454, 25 Sep. 2014, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETINGDISORDERS AND DISEASES USING CELL PENETRATION PEPTIDES (CPP); U.S.application 62/055,460, 25 Sep. 2014, MULTIFUNCTIONAL-CRISPR COMPLEXESAND/OR OPTIMIZED ENZYME LINKED FUNCTIONAL-CRISPR COMPLEXES; U.S.application 62/087,475, 4 Dec. 2014, FUNCTIONAL SCREENING WITH OPTIMIZEDFUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application 62/055,487, 25 Sep.2014, FUNCTIONAL SCREENING WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS;U.S. application 62/087,546, 4 Dec. 2014, MULTIFUNCTIONAL CRISPRCOMPLEXES AND/OR OPTIMIZED ENZYME LINKED FUNCTIONAL-CRISPR COMPLEXES;and U.S. application 62/098,285, 30 Dec. 2014, CRISPR MEDIATED IN VIVOMODELING AND GENETIC SCREENING OF TUMOR GROWTH AND METASTASIS.

Each of these patents, patent publications, and applications, and alldocuments cited therein or during their prosecution (“appin citeddocuments”) and all documents cited or referenced in the appln citeddocuments, together with any instructions, descriptions, productspecifications, and product sheets for any products mentioned therein orin any document therein and incorporated by reference herein, are herebyincorporated herein by reference, and may be employed in the practice ofthe invention. All documents (e.g., these patents, patent publicationsand applications and the appln cited documents) are incorporated hereinby reference to the same extent as if each individual document wasspecifically and individually indicated to be incorporated by reference.

Also with respect to general information on CRISPR-Cas Systems, mentionis made of the following (also hereby incorporated herein by reference):

-   -   Multiplex genome engineering using CRISPR/Cas systems. Cong, L.,        Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.        D., Wu, X., Jiang, W., Marraffini, L. A., & Zhang, F. Science        February 15; 339(6121):819-23 (2013);    -   RNA-guided editing of bacterial genomes using CRISPR-Cas        systems. Jiang W., Bikard D., Cox D., Zhang F, Marraffini L A.        Nat Biotechnol March; 31(3):233-9 (2013);    -   One-Step Generation of Mice Carrying Mutations in Multiple Genes        by CRISPR/Cas-Mediated Genome Engineering. Wang H., Yang H.,        Shivalila C S., Dawlaty M M., Cheng A W., Zhang F., Jaenisch R.        Cell May 9; 153(4):910-8 (2013);    -   Optical control of mammalian endogenous transcription and        epigenetic states. Konermann S, Brigham M D, Trevino A E, Hsu P        D, Heidenreich M, Cong L, Platt R J, Scott D A, Church G M,        Zhang F. Nature. August 22; 500(7463):472-6. doi:        10.1038/Nature12466. Epub 2013 Aug. 23 (2013);    -   Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome        Editing Specificity. Ran, F A., Hsu, P D., Lin, C Y.,        Gootenberg, J S., Konermann, S., Trevino, A E., Scott, D A.,        Inoue, A., Matoba, S., Zhang, Y., & Zhang, F. Cell August 28.        pii: S0092-8674(13)01015-5 (2013-A);    -   DNA targeting specificity of RNA-guided Cas9 nucleases. Hsu, P.,        Scott, D., Weinstein, J., Ran, F A., Konermann, S., Agarwala,        V., Li, Y., Fine, E., Wu, X., Shalem, O., Cradick, T J.,        Marraffini, L A., Bao, G., & Zhang, F. Nat Biotechnol        doi:10.1038/nbt.2647 (2013);    -   Genome engineering using the CRISPR-Cas9 system. Ran, F A., Hsu,        P D., Wright, J., Agarwala, V., Scott, D A., Zhang, F. Nature        Protocols November; 8(11):2281-308 (2013-B);    -   Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells.        Shalem, O., Sanjana, N E., Hartenian, E., Shi, X., Scott, D A.,        Mikkelson, T., Heckl, D., Ebert, B L., Root, D E., Doench, J G.,        Zhang, F. Science Dec. 12, 2013). [Epub ahead of print];    -   Crystal structure of cas9 in complex with guide RNA and target        DNA. Nishimasu, H., Ran, F A., Hsu, P D., Konermann, S.,        Shehata, S I., Dohmae, N., Ishitani, R., Zhang, F., Nureki, O.        Cell February 27, 156(5):935-49 (2014);    -   Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian        cells. Wu X., Scott D A., Kriz A J., Chiu A C., Hsu P D., Dadon        D B., Cheng A W., Trevino A E., Konermann S., Chen S., Jaenisch        R., Zhang F., Sharp P A. Nat Biotechnol. April 20. doi:        10.1038/nbt.2889 (2014);    -   CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling.        Platt R J, Chen S, Zhou Y, Yim M J, Swiech L, Kempton H R,        Dahlman J E, Parnas O, Eisenhaure T M, Jovanovic M, Graham D B,        Jhunjhunwala S, Heidenreich M, Xavier R J, Langer R, Anderson D        G, Hacohen N, Regev A, Feng G, Sharp P A, Zhang F. Cell 159(2):        440-455 DOI: 10.1016/j.ce11.2014.09.014(2014);    -   Development and Applications of CRISPR-Cas9 for Genome        Engineering, Hsu P D, Lander E S, Zhang F., Cell. June 5;        157(6):1262-78 (2014).    -   Genetic screens in human cells using the CRISPR/Cas9 system,        Wang T, Wei J J, Sabatini D M, Lander E S., Science. January 3;        343(6166): 80-84. doi:10.1126/science. 1246981 (2014);    -   Rational design of highly active sgRNAs for CRISPR-Cas9-mediated        gene inactivation, Doench J G, Hartenian E, Graham D B, Tothova        Z, Hegde M, Smith I, Sullender M, Ebert B L, Xavier R J, Root D        E., (published online 3 Sep. 2014) Nat Biotechnol. December;        32(12):1262-7 (2014);    -   In vivo interrogation of gene function in the mammalian brain        using CRISPR-Cas9, Swiech L, Heidenreich M, Banerjee A, Habib N,        Li Y, Trombetta J, Sur M, Zhang F., (published online 19        Oct. 2014) Nat Biotechnol. January; 33(1):102-6 (2015);    -   Genome-scale transcriptional activation by an engineered        CRISPR-Cas9 complex, Konermann S, Brigham M D, Trevino A E,        Joung J, Abudayyeh O O, Barcena C, Hsu P D, Habib N, Gootenberg        J S, Nishimasu H, Nureki O, Zhang F., Nature. January 29;        517(7536):583-8 (2015).    -   A split-Cas9 architecture for inducible genome editing and        transcription modulation, Zetsche B, Volz S E, Zhang F.,        (published online 2 Feb. 2015) Nat Biotechnol. February;        33(2):139-42 (2015);    -   Genome-wide CRISPR Screen in a Mouse Model of Tumor Growth and        Metastasis, Chen S, Sanjana N E, Zheng K, Shalem O, Lee K, Shi        X, Scott D A, Song J, Pan J Q, Weissleder R, Lee H, Zhang F,        Sharp P A. Cell 160, 1246-1260, Mar. 12, 2015 (multiplex screen        in mouse), and    -   In vivo genome editing using Staphylococcus aureus Cas9, Ran F        A, Cong L, Yan W X, Scott D A, Gootenberg J S, Kriz A J, Zetsche        B, Shalem O, Wu X, Makarova K S, Koonin E V, Sharp P A, Zhang        F., (published online 1 Apr. 2015), Nature. April 9;        520(7546):186-91 (2015).    -   Shalem et al., “High-throughput functional genomics using        CRISPR-Cas9,” Nature Reviews Genetics 16, 299-311 (May 2015).    -   Xu et al., “Sequence determinants of improved CRISPR sgRNA        design,” Genome Research 25, 1147-1157 (August 2015).    -   Parnas et al., “A Genome-wide CRISPR Screen in Primary Immune        Cells to Dissect Regulatory Networks,” Cell 162, 675-686 (Jul.        30, 2015).    -   Ramanan et al., CRISPR/Cas9 cleavage of viral DNA efficiently        suppresses hepatitis B virus,” Scientific Reports 5:10833. doi:        10.1038/srep10833 (Jun. 2, 2015)    -   Nishimasu et al., “Crystal Structure of Staphylococcus aureus        Cas9,” Cell 162, 1113-1126 (Aug. 27, 2015)    -   Zetsche et al. (2015), “Cpf1 is a single RNA-guided endonuclease        of a class 2 CRISPR-Cas system,” Cell 163, 759-771 (Oct.        22, 2015) doi: 10.1016/j.ce11.2015.09.038. Epub Sep. 25, 2015    -   Shmakov et al. (2015), “Discovery and Functional        Characterization of Diverse Class 2 CRISPR-Cas Systems,”        Molecular Cell 60, 385-397 (Nov. 5, 2015) doi:        10.1016/j.molce1.2015.10.008. Epub Oct. 22, 2015    -   Dahlman et al., “Orthogonal gene control with a catalytically        active Cas9 nuclease,” Nature Biotechnology 33, 1159-1161        (November, 2015)    -   Gao et al, “Engineered Cpf1 Enzymes with Altered PAM        Specificities,” bioRxiv 091611; doi:        http://dx.doi.org/10.1101/091611 Epub Dec. 4, 2016    -   Smargon et al. (2017), “Cas13b Is a Type VI-B CRISPR-Associated        RNA-Guided RNase Differentially Regulated by Accessory Proteins        Csx27 and Csx28,”Molecular Cell 65, 618-630 (Feb. 16, 2017) doi:        10.1016/j.molce1.2016.12.023. Epub Jan. 5, 2017        each of which is incorporated herein by reference, may be        considered in the practice of the instant invention, and        discussed briefly below:    -   Cong et al. engineered type II CRISPR-Cas systems for use in        eukaryotic cells based on both Streptococcus thermophilus Cas9        and also Streptococcus pyogenes Cas9 and demonstrated that Cas9        nucleases can be directed by short RNAs to induce precise        cleavage of DNA in human and mouse cells. Their study further        showed that Cas9 as converted into a nicking enzyme can be used        to facilitate homology-directed repair in eukaryotic cells with        minimal mutagenic activity. Additionally, their study        demonstrated that multiple guide sequences can be encoded into a        single CRISPR array to enable simultaneous editing of several at        endogenous genomic loci sites within the mammalian genome,        demonstrating easy programmability and wide applicability of the        RNA-guided nuclease technology. This ability to use RNA to        program sequence specific DNA cleavage in cells defined a new        class of genome engineering tools. These studies further showed        that other CRISPR loci are likely to be transplantable into        mammalian cells and can also mediate mammalian genome cleavage.        Importantly, it can be envisaged that several aspects of the        CRISPR-Cas system can be further improved to increase its        efficiency and versatility.    -   Jiang et al. used the clustered, regularly interspaced, short        palindromic repeats (CRISPR)-associated Cas9 endonuclease        complexed with dual-RNAs to introduce precise mutations in the        genomes of Streptococcus pneumoniae and Escherichia coli. The        approach relied on dual-RNA:Cas9-directed cleavage at the        targeted genomic site to kill unmutated cells and circumvents        the need for selectable markers or counter-selection systems.        The study reported reprogramming dual-RNA:Cas9 specificity by        changing the sequence of short CRISPR RNA (crRNA) to make        single- and multinucleotide changes carried on editing        templates. The study showed that simultaneous use of two crRNAs        enabled multiplex mutagenesis. Furthermore, when the approach        was used in combination with recombineering, in S. pneumoniae,        nearly 100% of cells that were recovered using the described        approach contained the desired mutation, and in E. coli, 65%        that were recovered contained the mutation.    -   Wang et al. (2013) used the CRISPR/Cas system for the one-step        generation of mice carrying mutations in multiple genes which        were traditionally generated in multiple steps by sequential        recombination in embryonic stem cells and/or time-consuming        intercrossing of mice with a single mutation. The CRISPR/Cas        system will greatly accelerate the in vivo study of functionally        redundant genes and of epistatic gene interactions.    -   Konermann et al. (2013) addressed the need in the art for        versatile and robust technologies that enable optical and        chemical modulation of DNA-binding domains based CRISPR Cas9        enzyme and also Transcriptional Activator Like Effectors    -   Ran et al. (2013-A) described an approach that combined a Cas9        nickase mutant with paired guide RNAs to introduce targeted        double-strand breaks. This addresses the issue of the Cas9        nuclease from the microbial CRISPR-Cas system being targeted to        specific genomic loci by a guide sequence, which can tolerate        certain mismatches to the DNA target and thereby promote        undesired off-target mutagenesis. Because individual nicks in        the genome are repaired with high fidelity, simultaneous nicking        via appropriately offset guide RNAs is required for        double-stranded breaks and extends the number of specifically        recognized bases for target cleavage. The authors demonstrated        that using paired nicking can reduce off-target activity by 50-        to 1,500-fold in cell lines and to facilitate gene knockout in        mouse zygotes without sacrificing on-target cleavage efficiency.        This versatile strategy enables a wide variety of genome editing        applications that require high specificity.    -   Hsu et al. (2013) characterized SpCas9 targeting specificity in        human cells to inform the selection of target sites and avoid        off-target effects. The study evaluated >700 guide RNA variants        and SpCas9-induced indel mutation levels at >100 predicted        genomic off-target loci in 293T and 293FT cells. The authors        that SpCas9 tolerates mismatches between guide RNA and target        DNA at different positions in a sequence-dependent manner,        sensitive to the number, position and distribution of        mismatches. The authors further showed that SpCas9-mediated        cleavage is unaffected by DNA methylation and that the dosage of        SpCas9 and sgRNA can be titrated to minimize off-target        modification. Additionally, to facilitate mammalian genome        engineering applications, the authors reported providing a        web-based software tool to guide the selection and validation of        target sequences as well as off-target analyses.    -   Ran et al. (2013-B) described a set of tools for Cas9-mediated        genome editing via non-homologous end joining (NHEJ) or        homology-directed repair (HDR) in mammalian cells, as well as        generation of modified cell lines for downstream functional        studies. To minimize off-target cleavage, the authors further        described a double-nicking strategy using the Cas9 nickase        mutant with paired guide RNAs. The protocol provided by the        authors experimentally derived guidelines for the selection of        target sites, evaluation of cleavage efficiency and analysis of        off-target activity. The studies showed that beginning with        target design, gene modifications can be achieved within as        little as 1-2 weeks, and modified clonal cell lines can be        derived within 2-3 weeks.    -   Shalem et al. described a new way to interrogate gene function        on a genome-wide scale. Their studies showed that delivery of a        genome-scale CRISPR-Cas9 knockout (GeCKO) library targeted        18,080 genes with 64,751 unique guide sequences enabled both        negative and positive selection screening in human cells. First,        the authors showed use of the GeCKO library to identify genes        essential for cell viability in cancer and pluripotent stem        cells. Next, in a melanoma model, the authors screened for genes        whose loss is involved in resistance to vemurafenib, a        therapeutic that inhibits mutant protein kinase BRAF. Their        studies showed that the highest-ranking candidates included        previously validated genes NF1 and MED12 as well as novel hits        NF2, CUL3, TADA2B, and TADA1. The authors observed a high level        of consistency between independent guide RNAs targeting the same        gene and a high rate of hit confirmation, and thus demonstrated        the promise of genome-scale screening with Cas9.    -   Nishimasu et al. reported the crystal structure of Streptococcus        pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 A°        resolution. The structure revealed a bilobed architecture        composed of target recognition and nuclease lobes, accommodating        the sgRNA:DNA heteroduplex in a positively charged groove at        their interface. Whereas the recognition lobe is essential for        binding sgRNA and DNA, the nuclease lobe contains the HNH and        RuvC nuclease domains, which are properly positioned for        cleavage of the complementary and non-complementary strands of        the target DNA, respectively. The nuclease lobe also contains a        carboxyl-terminal domain responsible for the interaction with        the protospacer adjacent motif (PAM). This high-resolution        structure and accompanying functional analyses have revealed the        molecular mechanism of RNA-guided DNA targeting by Cas9, thus        paving the way for the rational design of new, versatile        genome-editing technologies.    -   Wu et al. mapped genome-wide binding sites of a catalytically        inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with        single guide RNAs (sgRNAs) in mouse embryonic stem cells        (mESCs). The authors showed that each of the four sgRNAs tested        targets dCas9 to between tens and thousands of genomic sites,        frequently characterized by a 5-nucleotide seed region in the        sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin        inaccessibility decreases dCas9 binding to other sites with        matching seed sequences; thus 70% of off-target sites are        associated with genes. The authors showed that targeted        sequencing of 295 dCas9 binding sites in mESCs transfected with        catalytically active Cas9 identified only one site mutated above        background levels. The authors proposed a two-state model for        Cas9 binding and cleavage, in which a seed match triggers        binding but extensive pairing with target DNA is required for        cleavage.    -   Platt et al. established a Cre-dependent Cas9 knockin mouse. The        authors demonstrated in vivo as well as ex vivo genome editing        using adeno-associated virus (AAV)-, lentivirus-, or        particle-mediated delivery of guide RNA in neurons, immune        cells, and endothelial cells.    -   Hsu et al. (2014) is a review article that discusses generally        CRISPR-Cas9 history from yogurt to genome editing, including        genetic screening of cells.    -   Wang et al. (2014) relates to a pooled, loss-of-function genetic        screening approach suitable for both positive and negative        selection that uses a genome-scale lentiviral single guide RNA        (sgRNA) library.    -   Doench et al. created a pool of sgRNAs, tiling across all        possible target sites of a panel of six endogenous mouse and        three endogenous human genes and quantitatively assessed their        ability to produce null alleles of their target gene by antibody        staining and flow cytometry. The authors showed that        optimization of the PAM improved activity and also provided an        on-line tool for designing sgRNAs.    -   Swiech et al. demonstrate that AAV-mediated SpCas9 genome        editing can enable reverse genetic studies of gene function in        the brain.    -   Konermann et al. (2015) discusses the ability to attach multiple        effector domains, e.g., transcriptional activator, functional        and epigenomic regulators at appropriate positions on the guide        such as stem or tetraloop with and without linkers.    -   Zetsche et al. demonstrates that the Cas9 enzyme can be split        into two and hence the assembly of Cas9 for activation can be        controlled.    -   Chen et al. relates to multiplex screening by demonstrating that        a genome-wide in vivo CRISPR-Cas9 screen in mice reveals genes        regulating lung metastasis.    -   Ran et al. (2015) relates to SaCas9 and its ability to edit        genomes and demonstrates that one cannot extrapolate from        biochemical assays. Shalem et al. (2015) described ways in which        catalytically inactive Cas9 (dCas9) fusions are used to        synthetically repress (CRISPRi) or activate (CRISPRa)        expression, showing. advances using Cas9 for genome-scale        screens, including arrayed and pooled screens, knockout        approaches that inactivate genomic loci and strategies that        modulate transcriptional activity.    -   Shalem et al. (2015) described ways in which catalytically        inactive Cas9 (dCas9) fusions are used to synthetically repress        (CRISPRi) or activate (CRISPRa) expression, showing. advances        using Cas9 for genome-scale screens, including arrayed and        pooled screens, knockout approaches that inactivate genomic loci        and strategies that modulate transcriptional activity.    -   Xu et al. (2015) assessed the DNA sequence features that        contribute to single guide RNA (sgRNA) efficiency in        CRISPR-based screens. The authors explored efficiency of        CRISPR/Cas9 knockout and nucleotide preference at the cleavage        site. The authors also found that the sequence preference for        CRISPRi/a is substantially different from that for CRISPR/Cas9        knockout.    -   Parnas et al. (2015) introduced genome-wide pooled CRISPR-Cas9        libraries into dendritic cells (DCs) to identify genes that        control the induction of tumor necrosis factor (Tnf) by        bacterial lipopolysaccharide (LPS). Known regulators of Tlr4        signaling and previously unknown candidates were identified and        classified into three functional modules with distinct effects        on the canonical responses to LPS.    -   Ramanan et al (2015) demonstrated cleavage of viral episomal DNA        (cccDNA) in infected cells. The HBV genome exists in the nuclei        of infected hepatocytes as a 3.2 kb double-stranded episomal DNA        species called covalently closed circular DNA (cccDNA), which is        a key component in the HBV life cycle whose replication is not        inhibited by current therapies. The authors showed that sgRNAs        specifically targeting highly conserved regions of HBV robustly        suppresses viral replication and depleted cccDNA.    -   Nishimasu et al. (2015) reported the crystal structures of        SaCas9 in complex with a single guide RNA (sgRNA) and its        double-stranded DNA targets, containing the 5′-TTGAAT-3′ PAM and        the 5′-TTGGGT-3′ PAM. A structural comparison of SaCas9 with        SpCas9 highlighted both structural conservation and divergence,        explaining their distinct PAM specificities and orthologous        sgRNA recognition.    -   Zetsche et al. (2015) reported the characterization of Cpf1, a        putative class 2 CRISPR effector. It was demonstrated that Cpf1        mediates robust DNA interference with features distinct from        Cas9. Identifying this mechanism of interference broadens our        understanding of CRISPR-Cas systems and advances their genome        editing applications.    -   Shmakov et al. (2015) reported the characterization of three        distinct Class 2 CRISPR-Cas systems. The effectors of two of the        identified systems, C2c1 and C2c3, contain RuvC like        endonuclease domains distantly related to Cpf1. The third        system, Cas13b, contains an effector with two predicted HEPN        RNase domains.    -   Gao et al. (2016) reported using a structure-guided saturation        mutagenesis screen to increase the targeting range of Cpf1.        AsCpf1 variants were engineered with the mutations S542R/K607R        and S542R/K548V/N552R that can cleave target sites with        TYCV/CCCC and TATV PAMs, respectively, with enhanced activities        in vitro and in human cells.

Also, “Dimeric CRISPR RNA-guided Fok1 nucleases for highly specificgenome editing”, Shengdar Q. Tsai, Nicolas Wyvekens, Cyd Khayter,Jennifer A. Foden, Vishal Thapar, Deepak Reyon, Mathew J. Goodwin,Martin J. Aryee, J. Keith Joung Nature Biotechnology 32(6): 569-77(2014), relates to dimeric RNA-guided Fok1 Nucleases that recognizeextended sequences and can edit endogenous genes with high efficienciesin human cells.

In addition, mention is made of PCT application PCT/US14/70057, AttorneyReference 47627.99.2060 and BI-2013/107 entitled “DELIVERY, USE ANDTHERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FORTARGETING DISORDERS AND DISEASES USING PARTICLE DELIVERY COMPONENTS(claiming priority from one or more or all of US provisional patentapplications: 62/054,490, filed Sep. 24, 2014; 62/010,441, filed Jun.10, 2014; and 61/915,118, 61/915,215 and 61/915,148, each filed on Dec.12, 2013) (“the Particle Delivery PCT”), incorporated herein byreference, with respect to a method of preparing an sgRNA-and-Cas9protein containing particle comprising admixing a mixture comprising ansgRNA and Cas9 protein (and optionally HDR template) with a mixturecomprising or consisting essentially of or consisting of surfactant,phospholipid, biodegradable polymer, lipoprotein and alcohol; andparticles from such a process. For example, wherein Cas9 protein andsgRNA were mixed together at a suitable, e.g., 3:1 to 1:3 or 2:1 to 1:2or 1:1 molar ratio, at a suitable temperature, e.g., 15-30C, e.g.,20-25C, e.g., room temperature, for a suitable time, e.g., 15-45, suchas 30 minutes, advantageously in sterile, nuclease free buffer, e.g.,1×PBS. Separately, particle components such as or comprising: asurfactant, e.g., cationic lipid, e.g.,1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); phospholipid, e.g.,dimyristoylphosphatidylcholine (DMPC); biodegradable polymer, such as anethylene-glycol polymer or PEG, and a lipoprotein, such as a low-densitylipoprotein, e.g., cholesterol were dissolved in an alcohol,advantageously a C1-6 alkyl alcohol, such as methanol, ethanol,isopropanol, e.g., 100% ethanol. The two solutions were mixed togetherto form particles containing the Cas9-sgRNA complexes. Accordingly,sgRNA may be pre-complexed with the Cas9 protein, before formulating theentire complex in a particle. Formulations may be made with a differentmolar ratio of different components known to promote delivery of nucleicacids into cells (e.g. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC), polyethyleneglycol (PEG), and cholesterol) For example DOTAP:DMPC:PEG:CholesterolMolar Ratios may be DOTAP 100, DMPC 0, PEG 0, Cholesterol 0; or DOTAP90, DMPC 0, PEG 10, Cholesterol 0; or DOTAP 90, DMPC 0, PEG 5,Cholesterol 5. DOTAP 100, DMPC 0, PEG 0, Cholesterol 0. That applicationaccordingly comprehends admixing sgRNA, Cas9 protein and components thatform a particle; as well as particles from such admixing. Aspects of theinstant invention can involve particles; for example, particles using aprocess analogous to that of the Particle Delivery PCT, e.g., byadmixing a mixture comprising sgRNA and/or Cas9 as in the instantinvention and components that form a particle, e.g., as in the ParticleDelivery PCT, to form a particle and particles from such admixing (or,of course, other particles involving sgRNA and/or Cas9 as in the instantinvention).

EXAMPLES Example 1: Cas13b Orthologs

Cas13b proteins shown in Table 1 below are advantageously produced fromconstructs which are codon optimized for expression in mammalian cells.The sequences below are also given in FIG. 1, along with the accessionnumber of the protein sequence. PFS motifs of the Cas13b orthologs areprovided in Table 2.

TABLE 1 Bergeyella 1 MENKTSLGNNIYYNPFKPQDKSYFAGYFNAAMENTDSVFRELGzoohelcum KRLKGKEYTSENFFDAIFKENISLVEYERYVKLLSDYFPMARLLDKKEVPIKERKENFKKNFKGIIKAVRDLRNFYTHKEHGEVEITDEIFGVLDEMLKSTVLTVKKKKVKTDKTKEILKKSIEKQLDILCQKKLEYLRDTARKIEEKRRNQRERGEKELVAPFKYSDKRDDLIAAIYNDAFDVYIDKKKDSLKESSKAKYNTKSDPQQEEGDLKIPISKNGVVFLLSLFLTKQEIHAFKSKIAGFKATVIDEATVSEATVSHGKNSICFMATHEIFSHLAYKKLKRKVRTAEINYGEAENAEQLSVYAKETLMMQMLDELSKVPDVVYQNLSEDVQKTFIEDWNEYLKENNGDVGTMEEEQVIHPVIRKRYEDKFNYFAIRFLDEFAQFPTLRFQVEILGNYLHDSRPKENLISDRRIKEKITVFGRLSELEHKKALFIKNTETNEDREHYWEIFPNPNYDFPKENISVNDKDFPIAGSILDREKQPVAGKIGIKVKLLNQQYVSEVDKAVKAHQLKQRKASKPSIQNIIEEIVPINESNPKEAIVFGGQPTAYLSMNDIHSILYEFFDKWEKKKEKLEKKGEKELRKEIGKELEKKIVGKIQAQIQQIIDKDTNAKILKPYQDGNSTAIDKEKLIKDLKQEQNILQKLKDEQTVREKEYNDFIAYQDKNREINKVRDRNHKQYLKDYNLKRKYPEAPARKEVLYREKGKVAVWLANDIKRFMPTDFKNEWKGEQHSLLQKSLAYYEQCKEELKNLLPEKVFQHLPFKLGGYFQQKYLYQFYTCYLDKRLEYISGLVQQAENFKSENKVFKKVENECFKFLKKQNYTHKELDARVQSILGYPIFLERGFMDEKPTIIKGKTFKGNEALFADWFRYYKEYQNFQTFYDTENYPLVELEKKQADRKRKTKIYQQKKNDVFTLLMAKHIFKSVFKQDSIDQFSLEDLYQSREERLGNQERARQTGERNTNYIWNKTVDLKLCDGKITVENVKLKNVGDFIKYEYDQRVQAFLKYEENIEWQAFLIKESKEEENYPYVVEREIEQYEKVRREELLKEVHLIEEYILEKVKDKEILKKGDNQNFKYYILNGLLKQLKNEDVESYKVFNLNTEPEDVNINQLKQEATDLEQKAFVLTYIRNKFAHNQLPKKEFWDYCQEKYGKIEK EKTYAEYFAEVFKKEKEALIKPrevotella 2 MEDDKKTTDSIRYELKDKHFWAAFLNLARHNVYITVNHINKIL intermediaEEGEINRDGYETTLKNTWNEIKDINKKDRLSKLIIKHFPFLEAATYRLNPTDTTKQKEEKQAEAQSLESLRKSFFVFIYKLRDLRNHYSHYKHSKSLERPKFEEGLLEKMYNIFNASIRLVKEDYQYNKDINPDEDFKHLDRTEEEFNYYFTKDNEGNITESGLLFFVSLFLEKKDAIWMQQKLRGFKDNRENKKKMTNEVFCRSRMLLPKLRLQSTQTQDWILLDMLNELIRCPKSLYERLREEDREKFRVPIEIADEDYDAEQEPFKNTLVRHQDRFPYFALRYFDYNEIFTNLRFQIDLGTYHFSTYKKQIGDYKESHHLTHKLYGFERIQEFTKQNRPDEWRKFVKTFNSFETSKEPYIPETTPHYHLENQKIGIRFRNDNDKIWPSLKTNSEKNEKSKYKLDKSFQAEAFLSVHELLPMMFYYLLLKTENTDNDNEIETKKKENKNDKQEKHKIEEIIENKITEIYALYDTFANGEIKSIDELEEYCKGKDIEIGHLPKQMIAILKDEHKVMATEAERKQEEMLVDVQKSLESLDNQINEEIENVERKNSSLKSGKIASWLVNDMMRFQPVQKDNEGKPLNNSKANSTEYQLLQRTLAFFGSEHERLAPYFKQTKLIESSNPHPFLKDTEWEKCNNILSFYRSYLEAKKNFLESLKPEDWEKNQYFLKLKEPKTKPKTLVQGWKNGFNLPRGIFTEPIRKWFMKHRENITVAELKRVGLVAKVIPLFFSEEYKDSVQPFYNYHFNVGNINKPDEKNFLNCEERRELLRKKKDEFKKMTDKEKEENPSYLEFKSWNKFERELRLVRNQDIVTWLLCMELFNKKKIKELNVEKIYLKNINTNTTKKEKNTEEKNGEEKNIKEKNNILNRIMPMRLPIKVYGRENFSKNKKKKIRRNTFFTVYIEEKGTKLLKQGNFKALERDRRLGGLFSFVKTPSKAESKSNTISKLRVEYELGEYQKARIEIIKDMLALEKTLIDKYNSLDTDNFNKMLTDWLELKGEPDKASFQNDVDLLIAVRNAFSHNQYPMRNRIAFANINPFSLSSANTSEEKGLGIANQLKDKTHKTIEKIIEI EKPIETKE Prevotella 3MQKQDKLFVDRKKNAIFAFPKYITIMENKEKPEPIYYELTDKH buccaeFWAAFLNLARHNVYTTINHINRRLEIAELKDDGYMMGIKGSWNEQAKKLDKKVRLRDLIMKHFPFLEAAAYEMTNSKSPNNKEQREKEQSEALSLNNLKNVLFIFLEKLQVLRNYYSHYKYSEESPKPIFETSLLKNMYKVFDANVRLVKRDYMHHENIDMQRDFTHLNRKKQVGRTKNIIDSPNFHYHFADKEGNMTIAGLLFFVSLFLDKKDAIWMQKKLKGFKDGRNLREQMTNEVFCRSRISLPKLKLENVQTKDWMQLDMLNELVRCPKSLYERLREKDRESFKVPFDIFSDDYNAEEEPFKNTLVRHQDRFPYFVLRYFDLNEIFEQLRFQIDLGTYHFSIYNKRIGDEDEVRHLTHHLYGFARIQDFAPQNQPEEWRKLVKDLDHFETSQEPYISKTAPHYHLENEKIGIKFCSAHNNLFPSLQTDKTCNGRSKFNLGTQFTAEAFLSVHELLPMMFYYLLLTKDYSRKESADKVEGIIRKEISNIYAIYDAFANNEINSIADLTRRLQNTNILQGHLPKQMISILKGRQKDMGKEAERKIGEMIDDTQRRLDLLCKQTNQKIRIGKRNAGLLKSGKIADWLVNDMMRFQPVQKDQNNIPINNSKANSTEYRMLQRALALFGSENFRLKAYFNQMNLVGNDNPHPFLAETQWEHQTNILSFYRNYLEARKKYLKGLKPQNWKQYQHFLILKVQKTNRNTLVTGWKNSFNLPRGIFTQPIREWFEKHNNSKRIYDQILSFDRVGFVAKAIPLYFAEEYKDNVQPFYDYPFNIGNRLKPKKRQFLDKKERVELWQKNKELFKNYPSEKKKTDLAYLDFLSWKKFERELRLIKNQDIVTWLMFKELFNMATVEGLKIGEIHLRDIDTNTANEESNNILNRIMPMKLPVKTYETDNKGNILKERPLATFYIEETETKVLKQGNFKALVKDRRLNGLFSFAETTDLNLEEHPISKLSVDLELIKYQTTRISIFEMTLGLEKKLIDKYSTLPTDSFRNMLERWLQCKANRPELKNYVNSLIAVRNAFSHNQYPMYDATLFAEVKKFTLFPSVDTKKIELNIAPQLLEIVGKAIK EIEKSENKN Porphyromonas 4MNTVPASENKGQSRTVEDDPQYFGLYLNLARENLIEVESHVRI gingivalisKFGKKKLNEESLKQSLLCDHLLSVDRWTKVYGHSRRYLPFLHYFDPDSQIEKDHDSKTGVDPDSAQRLIRELYSLLDFLRNDFSHNRLDGTTFEHLEVSPDISSFITGTYSLACGRAQSRFAVFFKPDDFVLAKNRKEQLISVADGKECLTVSGFAFFICLFLDREQASGMLSRIRGFKRTDENWARAVHETFCDLCIRHPHDRLESSNTKEALLLDMLNELNRCPRILYDMLPEEERAQFLPALDENSMNNLSENSLDEESRLLWDGSSDWAEALTKRIRHQDRFPYLMLRFIEEMDLLKGIRFRVDLGEIELDSYSKKVGRNGEYDRTITDHALAFGKLSDFQNEEEVSRMISGEASYPVRFSLFAPRYAIYDNKIGYCHTSDPVYPKSKTGEKRALSNPQSMGFISVHDLRKLLLMELLCEGSFSRMQSDFLRKANRILDETAEGKLQFSALFPEMRHRFIPPQNPKSKDRREKAETTLEKYKQEIKGRKDKLNSQLLSAFDMDQRQLPSRLLDEWMNIRPASHSVKLRTYVKQLNEDCRLRLRKFRKDGDGKARAIPLVGEMATFLSQDIVRMIISEETKKLITSAYYNEMQRSLAQYAGEENRRQFRAIVAELRLLDPSSGHPFLSATMETAHRYTEGFYKCYLEKKREWLAKIFYRPEQDENTKRRISVFFVPDGEARKLLPLLIRRRMKEQNDLQDWIRNKQAHPIDLPSHLFDSKVMELLKVKDGKKKWNEAFKDWWSTKYPDGMQPFYGLRRELNIHGKSVSYIPSDGKKFADCYTHLMEKTVRDKKRELRTAGKPVPPDLAADIKRSFHRAVNEREFMLRLVQEDDRLMLMAINKMMTDREEDILPGLKNIDSILDEENQFSLAVHAKVLEKEGEGGDNSLSLVPATIEIKSKRKDWSKYIRYRYDRRVPGLMSHFPEHKATLDEVKTLLGEYDRCRIKIFDWAFALEGAIMSDRDLKPYLHESSSREGKSGEHSTLVKMLVEKKGCLTPDESQYLILIRNKAAHNQFPCAAEMPLIYRDVSAKVGSIEGSSAKDLPEGSSLVDSLWKKYEMIIRKILPILDPEN RFFGKLLNNMSQPINDLBacteroides 5 MESIKNSQKSTGKTLQKDPPYFGLYLNMALLNVRKVENHIRKW pyogenesLGDVALLPEKSGFHSLLTTDNLSSAKWTRFYYKSRKFLPFLEMFDSDKKSYENRRETAECLDTIDRQKISSLLKEVYGKLQDIRNAFSHYHIDDQSVKHTALIISSEMHRFIENAYSFALQKTRARFTGVFVETDFLQAEEKGDNKKFFAIGGNEGIKLKDNALIFLICLFLDREEAFKFLSRATGFKSTKEKGFLAVRETFCALCCRQPHERLLSVNPREALLMDMLNELNRCPDILFEMLDEKDQKSFLPLLGEEEQAHILENSLNDELCEAIDDPFEMIASLSKRVRYKNRFPYLMLRYIEEKNLLPFIRFRIDLGCLELASYPKKMGEENNYERSVTDHAMAFGRLTDFHNEDAVLQQITKGITDEVRFSLYAPRYAIYNNKIGFVRTSGSDKISFPTLKKKGGEGHCVAYTLQNTKSFGFISIYDLRKILLLSFLDKDKAKNIVSGLLEQCEKHWKDLSENLFDAIRTELQKEFPVPLIRYTLPRSKGGKLVSSKLADKQEKYESEFERRKEKLTEILSEKDFDLSQIPRRMIDEWLNVLPTSREKKLKGYVETLKLDCRERLRVFEKREKGEHPLPPRIGEMATDLAKDIIRMVIDQGVKQRITSAYYSEIQRCLAQYAGDDNRRHLDSIIRELRLKDTKNGHPFLGKVLRPGLGHTEKLYQRYFEEKKEWLEATFYPAASPKRVPRFVNPPTGKQKELPLIIRNLMKERPEWRDWKQRKNSHPIDLPSQLFENEICRLLKDKIGKEPSGKLKWNEMFKLYWDKEFPNGMQRFYRCKRRVEVFDKVVEYEYSEEGGNYKKYYEALIDEVVRQKISSSKEKSKLQVEDLTLSVRRVFKRAINEKEYQLRLLCEDDRLLFMAVRDLYDWKEAQLDLDKIDNMLGEPVSVSQVIQLEGGQPDAVIKAECKLKDVSKLMRYCYDGRVKGLMPYFANHEATQEQVEMELRHYEDHRRRVFNWVFALEKSVLKNEKLRRFYEESQGGCEHRRCIDALRKASLVSEEEYEFLVHIRNKSAHNQFPDLEIGKLPPNVTSGFCECIWSKYKAIICRIIPFIDPERRFFGKLLEQK Alistipes sp. 6MSNEIGAFREHQFAYAPGNEKQEEATFATYFNLALSNVEGMMF ZOR0009GEVESNPDKIEKSLDTLPPAILRQIASFIWLSKEDHPDKAYSTEEVKVIVTDLVRRLCFYRNYFSHCFYLDTQYFYSDELVDTTAIGEKLPYNFHHFITNRLFRYSLPEITLFRWNEGERKYEILRDGLIFFCCLFLKRGQAERFLNELRFFKRTDEEGRIKRTIFTKYCTRESHKHIGIEEQDFLIFQDIIGDLNRVPKVCDGVVDLSKENERYIKNRETSNESDENKARYRLLIREKDKFPYYLMRYIVDFGVLPCITFKQNDYSTKEGRGQFHYQDAAVAQEERCYNFVVRNGNVYYSYMPQAQNVVRISELQGTISVEELRNMVYASINGKDVNKSVEQYLLYHLHLLYEKITISGQTIKEGRVDVEDYRPLLDKLLLRPASNGEELRRELRKLLPKRVCDLLSNRFDCSEGVSAVEKRLKAILLRHEQLLLSQNPALHIDKIKSVIDYLYLFFSDDEKFRQQPTEKAHRGLKDEEFQMYHYLVGDYDSHPLALWKELEASGRLKPEMRKLTSATSLHGLYMLCLKGTVEWCRKQLMSIGKGTAKVEAIADRVGLKLYDKLKEYTPEQLEREVKLVVMHGYAAAATPKPKAQAAIPSKLTELRFYSFLGKREMSFAAFIRQDKKAQKLWLRNFYTVENIKTLQKRQAAADAACKKLYNLVGEVERVHTNDKVLVLVAQRYRERLLNVGSKCAVTLDNPERQQKLADVYEVQNAWLSIRFDDLDFTLTHVNLSNLRKAYNLIPRKHILAFKEYLDNRVKQKLCEECRNVRRKEDLCTCCSPRYSNLTSWLKENHSESSIEREAATMMLLDVERKLLSFLLDERRKAIIEYGKFIPFSALVKECRLADAGLCGIRNDVLHDNVISYADAIGKLSAYFPKEASEAVEYIRRTKEVREQRREE LMANSSQ Prevotella sp. 7MSKECKKQRQEKKRRLQKANFSISLTGKHVFGAYFNMARTNFV MA2016KTINYILPIAGVRGNYSENQINKMLHALFLIQAGRNEELTTEQKQWEKKLRLNPEQQTKFQKLLFKHFPVLGPMMADVADHKAYLNKKKSTVQTEDETFAMLKGVSLADCLDIICLMADTLTECRNFYTHKDPYNKPSQLADQYLHQEMIAKKLDKVVVASRRILKDREGLSVNEVEFLTGIDHLHQEVLKDEFGNAKVKDGKVMKTFVEYDDFYFKISGKRLVNGYTVTTKDDKPVNVNTMLPALSDFGLLYFCVLFLSKPYAKLFIDEVRLFEYSPFDDKENMIMSEMLSIYRIRTPRLHKIDSHDSKATLAMDIFGELRRCPMELYNLLDKNAGQPFFHDEVKHPNSHTPDVSKRLRYDDRFPTLALRYIDETELFKRIRFQLQLGSFRYKFYDKENCIDGRVRVRRIQKEINGYGRMQEVADKRMDKWGDLIQKREERSVKLEHEELYINLDQFLEDTADSTPYVTDARRPYNIHANRIGLYWEDSQNPKQYKVFDENGMYIPELVVTEDKKAPIKMPAPRCALSVYDLPAMLFYEYLREQQDNEFPSAEQVIIEYEDDYRKFFKAVAEGKLKPFKRPKEFRDFLKKEYPKLRMADIPKKLQLFLCSHGLCYNNKPETVYERLDRLTLQHLEERELHIQNRLEHYQKDRDMIGNKDNQYGKKSFSDVRHGALARYLAQSMMEWQPTKLKDKEKGHDKLTGLNYNVLTAYLATYGHPQVPEEGFTPRTLEQVLINAHLIGGSNPHPFINKVLALGNRNIEELYLHYLEEELKHIRSRIQSLSSNPSDKALSALPFIHHDRMRYHERTSEEMMALAARYTTIQLPDGLFTPYILEILQKHYTENSDLQNALSQDVPVKLNPTCNAAYLITLFYQTVLKDNAQPFYLSDKTYTRNKDGEKAESFSFKRAYELFSVLNNNKKDTFPFEMIPLFLTSDEIQERLSAKLLDGDGNPVPEVGEKGKPATDSQGNTIWKRRIYSEVDDYAEKLTDRDMKISFKGEWEKLPRWKQDKIIKRRDETRRQMRDELLQRMPRYIRDIKDNERTLRRYKTQDMVLFLLAEKMFTNIISEQSSEFNWKQMRLSKVCNEAFLRQTLTFRVPVTVGETTIYVEQENMSLKNYGEFYRFLTDDRLMSLLNNIVETLKPNENGDLVIRHTDLMSELAAYDQYRSTIFMLIQSIENLIITNNAVLDDPDADGFWVREDLPKRNNFASLLELINQLNNVELTDDERKLLVAIRNAFSHNSYNIDFSLIKDVKHLPEVAKGILQHLQSMLGVEITK Riemerella 8MEKPLLPNVYTLKHKFFWGAFLNIARHNAFITICHINEQLGLK anatipestiferTPSNDDKIVDVVCETWNNILNNDHDLLKKSQLTELILKHFPFLTAMCYHPPKKEGKKKGHQKEQQKEKESEAQSQAEALNPSKLIEALEILVNQLHSLRNYYSHYKHKKPDAEKDIFKHLYKAFDASLRMVKEDYKAHFTVNLTRDFAHLNRKGKNKQDNPDFNRYRFEKDGFFTESGLLFFTNLFLDKRDAYWMLKKVSGFKASHKQREKMTTEVFCRSRILLPKLRLESRYDHNQMLLDMLSELSRCPKLLYEKLSEENKKHFQVEADGFLDEIEEEQNPFKDTLIRHQDRFPYFALRYLDLNESFKSIRFQVDLGTYHYCIYDKKIGDEQEKRHLTRTLLSFGRLQDFTEINRPQEWKALTKDLDYKETSNQPFISKTTPHYHITDNKIGFRLGTSKELYPSLEIKDGANRIAKYPYNSGFVAHAFISVHELLPLMFYQHLTGKSEDLLKETVRHIQRIYKDFEEERINTIEDLEKANQGRLPLGAFPKQMLGLLQNKQPDLSEKAKIKIEKLIAETKLLSHRLNTKLKSSPKLGKRREKLIKTGVLADWLVKDFMRFQPVAYDAQNQPIKSSKANSTEFWEIRRALALYGGEKNRLEGYEKQTNLIGNTNPHPELNKENWKACRNLVDEYQQYLEQREKELEAIKNQPWEPYQYCLLLKIPKENRKNLVKGWEQGGISLPRGLFTEAIRETLSEDLMLSKPIRKEIKKHGRVGFISRAITLYFKEKYQDKHQSFYNLSYKLEAKAPLLKREEHYEYWQQNKPQSPTESQRLELHTSDRWKDYLLYKRWQHLEKKLRLYRNQDVMLWLMTLELTKNHFKELNLNYHQLKLENLAVNVQEADAKLNPLNQTLPMVLPVKVYPATAFGEVQYHKTPIRTVYIREEHTKALKMGNEKALVKDRRLNGLESFIKEENDTQKHPISQLRLRRELEIYQSLRVDAFKETLSLEEKLLNKHTSLSSLENEFRALLEEWKKEYAASSMVTDEHIAFIASVRNAFCHNQYPFYKEALHAPIPLFTVAQPTTEEKDGLG IAEALLKVLREYCEIVKSQIPrevotella 9 MEDDKKTTGSISYELKDKHFWAAFLNLARHNVYITINHINKLL aurantiacaEIREIDNDEKVLDIKTLWQKGNKDLNQKARLRELMTKHFPFLETAIYTKNKEDKKEVKQEKQAEAQSLESLKDCLFLFLDKLQEARNYYSHYKYSEFSKEPEFEEGLLEKMYNIFGNNIQLVINDYQHNKDINPDEDEKHLDRKGQFKYSFADNEGNITESGLLEFVSLFLEKKDAIWMQQKLNGEKDNLENKKKMTHEVECRSRILMPKLRLESTQTQDWILLDMLNELIRCPKSLYERLQGDDREKEKVPFDPADEDYNAEQEPEKNTLIRHQDREPYFVLRYEDYNEIEKNLREQIDLGTYHFSIYKKLIGGQKEDRHLTHKLYGFERIQEFAKQNRPDEWKAIVKDLDTYETSNKRYISETTPHYHLENQKIGIRERNGNKEIWPSLKTNDENNEKSKYKLDKQYQAEAFLSVHELLPMMFYYLLLKKEKPNNDEINASIVEGFIKREIRNIFKLYDAFANGEINNIDDLEKYCADKGIPKRHLPKQMVAILYDEHKDMVKEAKRKQKEMVKDTKKLLATLEKQTQKEKEDDGRNVKLLKSGEIARWLVNDMMREQPVQKDNEGKPLNNSKANSTEYQMLQRSLALYNNEEKPTRYFRQVNLIESNNPHPFLKWTKWEECNNILTFYYSYLTKKIEFLNKLKPEDWKKNQYFLKLKEPKTNRETLVQGWKNGENLPRGIFTEPIREWFKRHQNNSKEYEKVEALDRVGLVTKVIPLEEKEEYEKDKEENEKEDTQKEINDCVQPEYNEPYNVGNIHKPKEKDELHREERIELWDKKKDKFKGYKEKIKSKKLTEKDKEEFRSYLEFQSWNKFERELRLVRNQDIVTWLLCKELIDKLKIDELNIEELKKLRLNNIDTDTAKKEKNNILNRVMPMELPVTVYEIDDSHKIVKDKPLHTIYIKEAETKLLKQGNFKALVKDRRLNGLFSFVKTNSEAESKRNPISKLRVEYELGEYQEARIETIQDMLALEEKLINKYKDLPTNKESEMLNSWLEGKDEADKARFQNDVDELIAVRNAFSHNQYPMHNKIEFANIKPFSLYTANNSEEKGLGIANQLKDKTKETTDKIKKIEK PIETKE Prevotella 10MEDKPFWAAFFNLARHNVYLTVNHINKLLDLEKLYDEGKHKEI saccharolyticaFEREDIFNISDDVMNDANSNGKKRKLDIKKIWDDLDTDLTRKYQLRELILKHFPFIQPAIIGAQTKERTTIDKDKRSTSTSNDSLKQTGEGDINDLLSLSNVKSMEERLLQILEQLRNYYSHVKHSKSATMPNEDEDLLNWMRYIEIDSVNKVKEDYSSNSVIDPNTSFSHLIYKDEQGKIKPCRYPFTSKDGSINAFGLLEFVSLFLEKQDSIWMQKKIPGFKKASENYMKMTNEVFCRNHILLPKIRLETVYDKDWMLLDMLNEVVRCPLSLYKRLTPAAQNKFKVPEKSSDNANRQEDDNPFSRILVRHQNRFPYEVLREEDLNEVFTTLRFQINLGCYHFAICKKQIGDKKEVHHLIRTLYGFSRLQNFTQNTRPEEWNTLVKTTEPSSGNDGKTVQGVPLPYISYTIPHYQIENEKIGIKIFDGDTAVDTDIWPSVSTEKQLNKPDKYTLTPGFKADVFLSVHELLPMMFYYQLLLCEGMLKTDAGNAVEKVLIDTRNAIFNLYDAFVQEKINTITDLENYLQDKPILIGHLPKQMIDLLKGHQRDMLKAVEQKKAMLIKDTERRLKLLDKQLKQETDVAAKNTGTLLKNGQIADWLVNDMMRFQPVKRDKEGNPINCSKANSTEYQMLQRAFAFYATDSCRLSRYFTQLHLIHSDNSHLFLSRFEYDKQPNLIAFYAAYLKAKLEFLNELQPQNWASDNYFLLLRAPKNDRQKLAEGWKNGFNLPRGLFTEKIKTWFNEHKTIVDISDCDIFKNRVGQVARLIPVFFDKKFKDHSQPFYRYDFNVGNVSKPTEANYLSKGKREELFKSYQNKFKNNIPAEKTKEYREYKNFSLWKKFERELRLIKNQDILIWLMCKNLFDEKIKPKKDILEPRIAVSYIKLDSLQTNTSTAGSLNALAKVVPMTLAIHIDSPKPKGKAGNNEKENKEFTVYIKEEGTKLLKWGNFKTLLADRRIKGLFSYIEHDDIDLKQHPLTKRRVDLELDLYQTCRIDIFQQTLGLEAQLLDKYSDLNTDNFYQMLIGWRKKEGIPRNIKEDTDFLKDVRNAFSHNQYPDSKKIAFRRIRKFNPKELILEEEEGLGIATQMYKEVEKVVNRIKRIELFD HMPREF9712_ 11MKDILTTDTTEKQNRFYSHKIADKYFFGGYFNLASNNIYEVFE 03108EVNKRNTFGKLAKRDNGNLKNYIIHVFKDELSISDFEKRVAIF [MyroidesASYFPILETVDKKSIKERNRTIDLTLSQRIRQFREMLISLVTA odoratimimusVDQLRNFYTHYHHSDIVIENKVLDFLNSSFVSTALHVKDKYLK CCUG 10230]TDKTKEFLKETIAAELDILIEAYKKKQIEKKNTRFKANKREDILNAIYNEAFWSFINDKDKDKDKETVVAKGADAYFEKNHHKSNDPDFALNISEKGIVYLLSFFLTNKEMDSLKANLTGFKGKVDRESGNSIKYMATQRIYSFHTYRGLKQKIRTSEEGVKETLLMQMIDELSKVPNVVYQHLSTTQQNSFIEDWNEYYKDYEDDVETDDLSRVIHPVIRKRYEDRFNYFAIRFLDEFFDFPTLRFQVHLGDYVHDRRTKQLGKVESDRIIKEKVTVFARLKDINSAKASYFHSLEEQDKEELDNKWTLFPNPSYDFPKEHTLQHQGEQKNAGKIGIYVKLRDTQYKEKAALEEARKSLNPKERSATKASKYDIITQIIEANDNVKSEKPLVFTGQPIAYLSMNDIHSMLFSLLTDNAELKKTPEEVEAKLIDQIGKQINEILSKDTDTKILKKYKDNDLKETDTDKITRDLARDKEEIEKLILEQKQRADDYNYTSSTKFNIDKSRKRKHLLFNAEKGKIGVWLANDIKRFMFKESKSKWKGYQHTELQKLFAYFDTSKSDLELILSNMVMVKDYPIELIDLVKKSRTLVDFLNKYLEARLEYIENVITRVKNSIGTPQFKTVRKECFTFLKKSNYTVVSLDKQVERILSMPLFIERGFMDDKPTMLEGKSYKQHKEKFADWFVHYKENSNYQNFYDTEVYEITTEDKREKAKVTKKIKQQQKNDVFTLMMVNYMLEEVLKLSSNDRLSLNELYQTKEERIVNKQVAKDTQERNKNYIWNKVVDLQLCDGLVHIDNVKLKDIGNFRKYENDSRVKEFLTYQSDIVWSAYLSNEVDSNKLYVIERQLDNYESIRSKELLKEVQEIECSVYNQVANKESLKQSGNENFKQYVLQGLLPIGMDVREMLILSTDVKFKKEEIIQLGQAGEVEQDLYSLIYIRNKFAHNQLPIKEFFDFCENNYRSISDNEYYAEYYMEIFRSIKEKYAN Prevotella 12MEDDKKTTDSIRYELKDKHFWAAFLNLARHNVYITVNHINKIL intermediaEEDEINRDGYENTLENSWNEIKDINKKDRLSKLIIKHFPFLEATTYRQNPTDTTKQKEEKQAEAQSLESLKKSFFVFIYKLRDLRNHYSHYKHSKSLERPKFEEDLQNKMYNIFDVSIQFVKEDYKHNTDINPKKDFKHLDRKRKGKFHYSFADNEGNITESGLLFFVSLFLEKKDAIWVQKKLEGFKCSNKSYQKMTNEVFCRSRMLLPKLRLESTQTQDWILLDMLNELIRCPKSLYERLQGVNRKKFYVSFDPADEDYDAEQEPFKNTLVRHQDRFPYFALRYFDYNEVFANLRFQIDLGTYHFSIYKKLIGGQKEDRHLTHKLYGFERIQEFDKQNRPDEWKAIVKDSDTFKKKEEKEEEKPYISETTPHYHLENKKIGIAFKNHNIWPSTQTELTNNKRKKYNLGTSIKAEAFLSVHELLPMMFYYLLLKTENTKNDNKVGGKKETKKQGKHKIEAIIESKIKDIYALYDAFANGEINSEDELKEYLKGKDIKIVHLPKQMIAILKNEHKDMAEKAEAKQEKMKLATENRLKTLDKQLKGKIQNGKRYNSAPKSGEIASWLVNDMMRFQPVQKDENGESLNNSKANSTEYQLLQRTLAFFGSEHERLAPYFKQTKLIESSNPHPFLNDTEWEKCSNILSFYRSYLKARKNFLESLKPEDWEKNQYFLMLKEPKTNRETLVQGWKNGFNLPRGFFTEPIRKWFMEHWKSIKVDDLKRVGLVAKVTPLFFSEKYKDSVQPFYNYPFNVGDVNKPKEEDFLHREERIELWDKKKDKFKGYKAKKKFKEMTDKEKEEHRSYLEFQSWNKFERELRLVRNQDIVTWLLCTELIDKLKIDELNIKELKKLRLKDINTDTAKKEKNNILNRVMPMELPVTVYKVNKGGYIIKNKPLHTIYIKEAETKLLKQGNFKALVKDRRLNGLFSFVKTPSEAESESNPISKLRVEYELGKYQNARLDIIEDMLALEKKLIDKYNSLDTDNFHNMLTGWLELKGEAKKARFQNDVKLLTAVRNAFSHNQYPMYDENLFGNIERFSLSSSNIIESKGLDIAAKLKEEVSKAAKKIQNEEDNKKEK ET Capnocytophaga 13MKNIQRLGKGNEFSPFKKEDKFYFGGFLNLANNNIEDFFKEII canimorsusTRFGIVITDENKKPKETFGEKILNEIFKKDISIVDYEKWVNIFADYFPFTKYLSLYLEEMQFKNRVICFRDVMKELLKTVEALRNFYTHYDHEPIKIEDRVFYFLDKVLLDVSLTVKNKYLKTDKTKEFLNQHIGEELKELCKQRKDYLVGKGKRIDKESEIINGIYNNAFKDFICKREKQDDKENHNSVEKILCNKEPQNKKQKSSATVWELCSKSSSKYTEKSFPNRENDKHCLEVPISQKGIVFLLSFFLNKGEIYALTSNIKGFKAKITKEEPVTYDKNSIRYMATHRMFSFLAYKGLKRKIRTSEINYNEDGQASSTYEKETLMLQMLDELNKVPDVVYQNLSEDVQKTFIEDWNEYLKENNGDVGTMEEEQVIHPVIRKRYEDKFNYFAIRFLDEFAQFPTLRFQVHLGNYLCDKRTKQICDTTTEREVKKKITVFGRLSELENKKAIFLNEREEIKGWEVFPNPSYDFPKENISVNYKDFPIVGSILDREKQPVSNKIGIRVKIADELQKREIDKAIKEKKLRNPKNRANQDEKQKERLVNEIVSTNSNEQGEPVVFIGQPTAYLSMNDIHSVLYEFLINKISGEALETKIVEKINSETQIKQIIGKDATTKILKPYTNANSINREKLLRDLEQEQQILKTLLEEQQQREKDKKDKKSKRKHELYPSEKGKVAVWLANDIKRFMPKAFKEQWRGYHHSLLQKYLAYYEQSKEELKNLLPKEVFKHFPFKLKGYFQQQYLNQFYTDYLKRRLSYVNELLLNIQNFKNDKDALKATEKECFKFFRKQNYIINPINIQIQSILVYPIFLKRGFLDEKPTMIDREKFKENKDTELADWFMHYKNYKEDNYQKFYAYPLEKVEEKEKFKRNKQINKQKKNDVYTLMMVEYIIQKIFGDKFVEENPLVLKGIFQSKAERQQNNTHAATTQERNLNGILNQPKDIKIQGKITVKGVKLKDIGNFRKYEIDQRVNTFLDYEPRKEWMAYLPNDWKEKEKQGQLPPNNVIDRQISKYETVRSKILLKDVQELEKIISDEIKEEHRHDLKQGKYYNFKYYILNGLLRQLKNENVENYKVFKLNTNPEKVNITQLKQEATDLEQKAFVLTYIRNKFAHNQLPKKEFWDYCQEKYGKIEKEKTYAEYFAEVFKREKEALIK Porphyromonas 14MTEQSERPYNGTYYTLEDKHFWAAFLNLARHNAYITLTHIDRQ gulaeLAYSKADITNDQDVLSFKALWKNFDNDLERKSRLRSLILKHFSFLEGAAYGKKLFESKSSGNKSSKNKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHSGSSELPLFDGNMLQRLYNVFDVSVQRVKIDHEHNDEVDPHYHFNHLVRKGKKDRYGHNDNPSFKHHFVDGEGMVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTETYQQMTNEVFCRSRISLPKLKLESLRMDDWMLLDMLNELVRCPKPLYDRLREDDRACFRVPVDILPDEDDTDGGGEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKMIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYISQTSPHYHIEKGKIGLRFMPEGQHLWPSPEVGTTRTGRSKYAQDKRLTAEAFLSVHELMPMMFYYFLLREKYSEEVSAERVQGRIKRVIEDVYAVYDAFARDEINTRDELDACLADKGIRRGHLPRQMIAILSQEHKDMEEKIRKKLQEMMADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDASGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLRARKAFLERIGRSDRVENRPFLLLKEPKTDRQTLVAGWKGEFHLPRGIFTEAVRDCLIEMGHDEVASYKEVGFMAKAVPLYFERACEDRVQPFYDSPFNVGNSLKPKKGRFLSKEERAEEWERGKERFRDLEAWSYSAARRIEDAFAGIEYASPGNKKKIEQLLRDLSLWEAFESKLKVRADRINLAKLKKEILEAQEHPYHDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRPNVQEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEEAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGGLAMEQYPISKLRVEYELAKYQTARVCVFELTLRLEESLLTRYPHLPDESFREMLESWSDPLLAKWPELHGKVRLLIAVRNAFSHNQYPMYDEAVFSSIRKYDPSSPDAIEERMGLNIAHRLSEE VKQAKETVERIIQAPrevotella sp. 15 MNIPALVENQKKYFGTYSVMAMLNAQTVLDHIQKVADIEGEQN P5-125ENNENLWFHPVMSHLYNAKNGYDKQPEKTMFIIERLQSYFPFLKIMAENQREYSNGKYKQNRVEVNSNDIFEVLKRAFGVLKMYRDLTNHYKTYEEKLNDGCEFLTSTEQPLSGMINNYYTVALRNMNERYGYKTEDLAFIQDKRFKFVKDAYGKKKSQVNTGFFLSLQDYNGDTQKKLHLSGVGIALLICLFLDKQYINIFLSRLPIFSSYNAQSEERRIIIRSFGINSIKLPKDRIHSEKSNKSVAMDMLNEVKRCPDELFTTLSAEKQSRFRIISDDHNEVLMKRSSDRFVPLLLQYIDYGKLFDHIRFHVNMGKLRYLLKADKTCIDGQTRVRVIEQPLNGFGRLEEAETMRKQENGTFGNSGIRIRDFENMKRDDANPANYPYIVDTYTHYILENNKVEMFINDKEDSAPLLPVIEDDRYVVKTIPSCRMSTLEIPAMAFHMFLFGSKKTEKLIVDVHNRYKRLFQAMQKEEVTAENIASFGIAESDLPQKILDLISGNAHGKDVDAFIRLTVDDMLTDTERRIKRFKDDRKSIRSADNKMGKRGFKQISTGKLADFLAKDIVLFQPSVNDGENKITGLNYRIMQSAIAVYDSGDDYEAKQQFKLMFEKARLIGKGTTEPHPFLYKVFARSIPANAVEFYERYLIERKFYLTGLSNEIKKGNRVDVPFIRRDQNKWKTPAMKTLGRIYSEDLPVELPRQMFDNEIKSHLKSLPQMEGIDFNNANVTYLIAEYMKRVLDDDFQTFYQWNRNYRYMDMLKGEYDRKGSLQHCFTSVEEREGLWKERASRTERYRKQASNKIRSNRQMRNASSEEIETILDKRLSNSRNEYQKSEKVIRRYRVQDALLFLLAKKTLTELADFDGERFKLKEIMPDAEKGILSEIMPMSFTFEKGGKKYTITSEGMKLKNYGDFFVLASDKRIGNLLELVGSDIVSKEDIMEEFNKYDQCRPEISSIVFNLEKWAFDTYPELSARVDREEKVDFKSILKILLNNKNINKEQSDILRKIRNAFDHNNYPDKGVVEIKALP EIAMSIKKAFGEYAIMKFlavobacterium 16 MENLNKILDKENEICISKIFNTKGIAAPITEKALDNIKSKQKNbranchiophilum DLNKEARLHYFSIGHSFKQIDTKKVFDYVLIEELKDEKPLKFITLQKDFFTKEFSIKLQKLINSIRNINNHYVHNFNDINLNKIDSNVFHFLKESFELAIIEKYYKVNKKYPLDNEIVLFLKELFIKDENTALLNYFTNLSKDEAIEYILTFTITENKIWNINNEHNILNIEKGKYLTFEAMLFLITIFLYKNEANHLLPKLYDFKNNKSKQELFTFFSKKFTSQDIDAEEGHLIKFRDMIQYLNHYPTAWNNDLKLESENKNKIMTTKLIDSIIEFELNSNYPSFATDIQFKKEAKAFLFASNKKRNQTSFSNKSYNEEIRHNPHIKQYRDEIASALTPISFNVKEDKFKIFVKKHVLEEYFPNSIGYEKFLEYNDFTEKEKEDFGLKLYSNPKTNKLIERIDNHKLVKSHGRNQDRFMDFSMRFLAENNYFGKDAFFKCYKFYDTQEQDEFLQSNENNDDVKFHKGKVTTYIKYEEHLKNYSYWDCPFVEENNSMSVKISIGSEEKILKIQRNLMIYFLENALYNENVENQGYKLVNNYYRELKKDVEESIASLDLIKSNPDFKSKYKKILPKRLLHNYAPAKQDKAPENAFETLLKKADFREEQYKKLLKKAEHEKNKEDFVKRNKGKQFKLHFIRKACQMMYFKEKYNTLKEGNAAFEKKDPVIEKRKNKEHEFGHHKNLNITREEFNDYCKWMFAFNGNDSYKKYLRDLFSEKHFFDNQEYKNLFESSVNLEAFYAKTKELFKKWIETNKPTNNENRYTLENYKNLILQKQVFINVYHFSKYLIDKNLLNSENNVIQYKSLENVEYLISDFYFQSKLSIDQYKTCGKLFNKLKSNKLEDCLLYEIAYNYIDKKNVHKIDIQKILTSKIILTINDANTPYKISVPFNKLERYTEMIAIKNQNNLKARFLIDLPLYLSKNKIKKGKDSAGYEIIIKNDLEIEDINTINNKIINDSVKFTEVLMELEKYFILKDKCILSKNYIDNSEIPSLKQFSKVWIKENENEIINYRNIACHFHLPLLETFDNLLLNVEQKFIKEELQNVSTINDLSKPQEYLILLFIKFKHNNFYLNLFNKNESKTIKNDKEVKKNRVLQKFINQVILKKK Myroides 17MKDILTTDTTEKQNRFYSHKIADKYFFGGYFNLASNNIYEVFE odoratimimusEVNKRNTFGKLAKRDNGNLKNYIIHVFKDELSISDFEKRVAIFASYFPILETVDKKSIKERNRTIDLTLSQRIRQFREMLISLVTAVDQLRNFYTHYHHSDIVIENKVLDFLNSSFVSTALHVKDKYLKTDKTKEFLKETIAAELDILIEAYKKKQIEKKNTRFKANKREDILNAIYNEAFWSFINDKDKDKDKETVVAKGADAYFEKNHHKSNDPDFALNISEKGIVYLLSFFLTNKEMDSLKANLTGFKGKVDRESGNSIKYMATQRIYSFHTYRGLKQKIRTSEEGVKETLLMQMIDELSKVPNVVYQHLSTTQQNSFIEDWNEYYKDYEDDVETDDLSRVTHPVIRKRYEDRFNYFAIRFLDEFFDFPTLRFQVHLGDYVHDRRTKQLGKVESDRIIKEKVTVFARLKDINSAKASYFHSLEEQDKEELDNKWTLFPNPSYDFPKEHTLQHQGEQKNAGKIGIYVKLRDTQYKEKAALEEARKSLNPKERSATKASKYDIITQIIEANDNVKSEKPLVFTGQPIAYLSMNDIHSMLFSLLTDNAELKKTPEEVEAKLIDQIGKQINEILSKDTDTKILKKYKDNDLKETDTDKITRDLARDKEEIEKLILEQKQRADDYNYTSSTKFNIDKSRKRKHLLFNAEKGKIGVWLANDIKRFMFKESKSKWKGYQHIELQKLFAYFDTSKSDLELILSNMVMVKDYPIELIDLVKKSRTLVDFLNKYLEARLEYIENVITRVKNSIGTPQFKTVRKECFTFLKKSNYTVVSLDKQVERILSMPLFIERGFMDDKPTMLEGKSYKQHKEKFADWFVHYKENSNYQNFYDTEVYEITTEDKREKAKVTKKIKQQQKNDVFTLMMVNYMLEEVLKLSSNDRLSLNELYQTKEERIVNKQVAKDTQERNKNYIWNKVVDLQLCDGLVHIDNVKLKDIGNFRKYENDSRVKEFLTYQSDIVWSAYLSNEVDSNKLYVIERQLDNYESIRSKELLKEVQEIECSVYNQVANKESLKQSGNENFKQYVLQGLLPIGMDVREMLILSTDVKFKKEEIIQLGQAGEVEQDLYSLIYIRNKFAHNQLPIKEFFDFCENNYRSISDNEYYAEYYMEIFRSIKEKYAN Flavobacterium 18MSSKNESYNKQKTFNHYKQEDKYFFGGFLNNADDNLRQVGKEF columnareKTRINFNHINNNELASVFKDYFNKEKSVAKREHALNLLSNYFPVLERIQKHTNHNFEQTREIFELLLDTIKKLRDYYTHHYHKPITINPKIYDFLDDTLLDVLITIKKKKVKNDTSRELLKEKLRPELTQLKNQKREELIKKGKKLLEENLENAVFNHCLIPFLEENKTDDKQNKTVSLRKYRKSKPNEETSITLTQSGLVFLMSFFLHRKEFQVFTSGLERFKAKVNTIKEEEISLNKNNIVYMITHWSYSYYNFKGLKHRIKTDQGVSTLEQNNTTHSLTNTNTKEALLTQIVDYLSKVPNEIYETLSEKQQKEFEEDINEYMRENPENEDSTFSSIVSHKVIRKRYENKFNYFAMRFLDEYAELPTLRFMVNFGDYIKDRQKKILESIQFDSERIIKKEIHLFEKLSLVTEYKKNVYLKETSNIDLSRFPLFPNPSYVMANNNIPFYIDSRSNNLDEYLNQKKKAQSQNKKRNLTFEKYNKEQSKDAIIAMLQKEIGVKDLQQRSTIGLLSCNELPSMLYEVIVKDIKGAELENKIAQKIREQYQSIRDFTLDSPQKDNIPTTLIKTINTDSSVTFENQPIDIPRLKNALQKELTLTQEKLLNVKEHEIEVDNYNRNKNTYKFKNQPKNKVDDKKLQRKYVFYRNEIRQEANWLASDLIHFMKNKSLWKGYMHNELQSFLAFFEDKKNDCIALLETVFNLKEDCILTKGLKNLFLKHGNFIDFYKEYLKLKEDFLSTESTFLENGFIGLPPKILKKELSKRLKYIFIVFQKRQFIIKELEEKKNNLYADAINLSRGIFDEKPTMIPFKKPNPDEFASWFVASYQYNNYQSFYELTPDIVERDKKKKYKNLRAINKVKIQDYYLKLMVDTLYQDLFNQPLDKSLSDFYVSKAEREKIKADAKAYQKLNDSSLWNKVIHLSLQNNRITANPKLKDIGKYKRALQDEEKIATLLTYDARTWTYALQKPEKNENDYKELHYTALNMELQEFYEKVRSKELLKQVQELEKKILDKYDFSNNASHPEDLEIEDKKGKRHPNFKLYITKALLKNESEIINLENIDIEILLKYYDYNTEELKEKIKNMDEDEKAKIINTKENYNKITNVLIKKALVLIIIRNKMAHNQYPPKFIYDLANRFVPKKEEEYFATYFNRVFETITKELW ENKEKKDKTQV Porphyromonas 19MTEQNEKPYNGTYYTLEDKHFWAAFLNLARHNAYITLAHIDRQ  gingivalisLAYSKADITNDEDILFFKGQWKNLDNDLERKARLRSLILKHFSFLEGAAYGKKLFESQSSGNKSSKKKELSKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHPESSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDKYGNNDNPFFKHHFVDREGTVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTEAYQQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKSLYDRLREEDRARFRVPVDILSDEDDTDGTEEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKNIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYITQTTPHYHIEKGKIGLRFVPEGQHLWPSPEVGATRTGRSKYAQDKRLTAEAFLSVHELMPMNIFYYFLLREKYSEEVSAEKVQGRIKRVIEDVYAVYDAFARDEINTRDELDACLADKGIRRGHLPRQMIAILSQEHKDMEEKVRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVVADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLEARKAFLQSIGRSDRVENHRFLLLKEPKTDRQTLVAGWKGEFHLPRGIFTEAVRDCLIEMGYDEVGSYKEVGFMAKAVPLYFERASKDRVQPFYDYPFNVGNSLKPKKGRFLSKEKRAEEWESGKERFRLAKLKKEILEAKEHPYHDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRTDVQEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEQAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGALAMEQYPISKLRVEYELAKYQTARVCAFEQTLELEESLLTRYPHLPDKNFRKMLESWSDPLLDKWPDLHGNVRLLIAVRNAFSHNQYPMYDETLFSSIRKYDPSSPDAIEERMGLNIAHRLSEEVKQAKEMVERII QA Porphyromonas 20MTEQSERPYNGTYYTLEDKHFWAAFLNLARHNAYITLTHIDRQ sp. COT-052LAYSKADITNDQDVLSFKALWKNFDNDLERKSRLRSLILKHFS OH4946FLEGAAYGKKLFESKSSGNKSSKNKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHSESSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDRYGHNDNPSFKHHFVDSEGMVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTETYQQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKPLYDRLREDDRACFRVPVDILPDEDDTDGGGEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKMIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYISQTTPHYHIEKGKIGLRFVPEGQHLWPSPEVGTTRTGRSKYAQDKRLTAEAFLSVHELMPMMFYYFLLREKYSEEVSAEKVQGRIKRVIEDVYAIYDAFARDEINTLKELDACLADKGIRRGHLPKQMIGILSQERKDMEEKVRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLRARKAFLERIGRSDRVENCPFLLLKEPKTDRQTLVAGWKGEFHLPRGIFTEAVRDCLIEMGYDEVGSYREVGFMAKAVPLYFERACEDRVQPFYDSPFNVGNSLKPKKGRFLSKEDRAEEWERGKERFRDLEAWSHSAARRIKDAFAGIEYASPGNKKKIEQLLRDLSLWEAFESKLKVRADKINLAKLKKEILEAQEHPYHDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRPNVQEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEEAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGGLAMEQYPISKLRVEYELAKYQTARVCVFELTLRLEESLLSRYPHLPDESFREMLESWSDPLLAKWPELHGKVRLLIAVRNAFSHNQYPMYDEAVFSSIRKYDPSSPDAIEERMGLNIAHRLSEE VKQAKETVERIIQA Prevotella 21MEDDKKTKESTNMLDNKHFWAAFLNLARHNVYITVNHINKVLE intermediaLKNKKDQDIIIDNDQDILAIKTHWEKVNGDLNKTERLRELMTKHFPFLETAIYTKNKEDKEEVKQEKQAKAQSFDSLKHCLFLFLEKLQEARNYYSHYKYSESTKEPMLEKELLKKMYNIFDDNIQLVIKDYQHNKDINPDEDFKHLDRTEEEFNYYFTTNKKGNITASGLLFFVSLFLEKKDAIWMQQKLRGFKDNRESKKKMTHEVFCRSRMLLPKLRLESTQTQDWILLDMLNELIRCPKSLYERLQGEYRKKFNVPFDSADEDYDAEQEPFKNTLVRHQDRFPYFALRYFDYNEIFTNLRFQIDLGTYHFSIYKKLIGGQKEDRHLTHKLYGFERIQEFAKQNRTDEWKAIVKDFDTYETSEEPYISETAPHYHLENQKIGIRFRNDNDEIWPSLKTNGENNEKRKYKLDKQYQAEAFLSVHELLPMMFYYLLLKKEEPNNDKKNASIVEGFIKREIRDIYKLYDAFANGEINNIDDLEKYCEDKGIPKRHLPKQMVAILYDEHKDMAEEAKRKQKEMVKDTKKLLATLEKQTQGEIEDGGRNIRLLKSGEIARWLVNDMMRFQPVQKDNEGNPLNNSKANSTEYQMLQRSLALYNKEEKPTRYFRQVNLINSSNPHPFLKWTKWEECNNILSFYRSYLTKKIEFLNKLKPEDWEKNQYFLKLKEPKTNRETLVQGWKNGFNLPRGIFTEPIREWFKRHQNDSEEYEKVETLDRVGLVTKVIPLFFKKEDSKDKEEYLKKDAQKEINNCVQPFYGFPYNVGNIHKPDEKDFLPSEERKKLWGDKKYKFKGYKAKVKSKKLTDKEKEEYRSYLEFQSWNKFERELRLVRNQDIVTWLLCTELIDKLKVEGLNVEELKKLRLKDIDTDTAKQEKNNILNRVMPMQLPVTVYEIDDSHNIVKDRPLHTVYIEETKTKLLKQGNFKALVKDRRLNGLFSFVDTSSETELKSNPISKSLVEYELGEYQNARIETIKDMLLLEETLIEKYKTLPTDNFSDMLNGWLEGKDEADKARFQNDVKLLVAVRNAFSHNQYPMRNRIAFANINPFSLSSADTSEEKKLDIANQLKDKTHKII KRIIEIEKPIETKE

TABLE 2 Acces- sory Ortholog No. protein 5′PFS 3′PFSBergeyella zoohelcum 1 A NGA Bergeyella zoohelcum 1 csx27 A NGAPrevotella intermedia 2 A NGA Prevotella intermedia 2 csx28 A NGAPrevotella buccae 3 A NGA Prevotella buccae 3 csx28 A NGABacteroides pyogenes 5 A NGA Alistipes sp. ZOR0009 6 TG NA(G)Prevotella sp. MA2016 7 AT Riemerella anatipestifer 8 A NGARiemerella anatipestifer 8 csx28 A NGA Prevotella aurantiaca 9 G NAAPrevotella aurantiaca 9 csx28 Prevotella 10 AG saccharolyticaPrevotella intermedia 12 AG Capnocytophaga 13 A NAA canimorsusCapnocytophaga 13 csx27 A NAA canimorsus Porphyromonas gulae 14 A NAAPorphyromonas gulae 14 csx28 A NGA Prevotella sp. P5-125 15 ATFlavobacterium 16 TG branchiophilum Flavobacterium 16 csx27 TAbranchiophilum Myroides odoratimimus 17 T NAA Porphyromonas gingivalis19 A NAA Porphyromonas gingivalis 19 csx28 A Prevotella intermedia 21 ANGA Prevotella intermedia 21 csx28 A NGA

Example 2: Activity of Cas13b in Mammalian Cells

HEK293T cells were transfected using the standard lipofectamine 2000protocol with the following plasmids:

(a) Cas13b (or control C2c2) mammalian expression plasmid, with a 1×Nuclear Export Sequence tag on the C-term of the gene.

(b) crRNA expression plasmid, each expressing either a targeting crRNAagainst Gaussia Luciferase or a non-targeting crRNA.

(c) Luciferase reporter plasmid, expressing Gaussia and Cypridinialuciferase from two separate promoters.

Gaussia luciferase was targeted for knockdown, and the level ofcypridinia luciferase was used to control for transfection efficiency.Leptotrichia wadeii C2c2 was used for comparison with C2c2 orthologs.

The following spacer sequences were used in the respective guides:

TABLE 3 Cas13b-guide 2 GGGCATTGGCTTCCATCTCTTTGAGCACCT (SEQ ID NO: 167Cas13b-guide NT GCAGGGTTTTCCCAGTCACGACGTTGTAAA (SEQ ID NO: 168)Experiment #2 (14 vs. 15 vs. C2c2) Cas13b-guide0GAAGTCTTCGTTGTTCTCGGTGGGCTTGGC (SEQ ID NO: 169 Cas13b-guide1GGGCATTGGCTTCCATCTCTTTGAGCACCT (SEQ ID NO: 170) Cas13b-guide2GACAGGCAGATCAGACAGCCCCTGGTGCAG (SEQ ID NO: 171) Cas13b-guide3GTAGGTGTGGCAGCGTCCTGGGATGAACTT (SEQ ID NO: 172) Cas13b-guide4GGAATGTCGACGATCGCCTCGCCTATGCCG (SEQ ID NO: 173) C2c2-guide0AGTCTTCGTTGTTCTCGGTGGGCTTGGC (SEQ ID NO: 174) C2c2-guide1GCATTGGCTTCCATCTCTTTGAGCACCT (SEQ ID NO: 175) C2c2-guide2CAGGCAGATCAGACAGCCCCTGGTGCAG (SEQ ID NO: 176) C2c2-guide3AGGTGTGGCAGCGTCCTGGGATGAACTT (SEQ ID NO: 177) C2c2-guide4AATGTCGACGATCGCCTCGCCTATGCCG (SEQ ID NO: 178) Cas13b-guide NTGCAGGGTTTTCCCAGTCACGACGTTGTAAA (SEQ ID NO: 179) C2c2-guide NTAGGGTTTTCCCAGTCACGACGTTGTAAA (SEQ ID NO: 180)

The results for the different Cas13b orthologs are provided in FIG. 2.The orthologs are classified as Low/No activity, Medium, High, or Gold.“Gold” orthologs provided >80% knockdown of luciferase activity with themajority of guides tested. “High” orthologs provided >50% knockdown ofluciferase activity with the majority of guides tested. “Medium”orthologs provided ˜50% knockdown of luciferase activity with themajority of guides tested. “Low/No” orthologs provided <80% knockdown ofluciferase activity with all of guides tested.

FIG. 3 shows normalised comparison data for the activity of several ofthe orthologs tested, using Guide 2 (GGGCATTGGCTTCCATCTCTTTGAGCACCT)(SEQ ID NO: 181) and a non-targeting guide as control(GCAGGGTTTTCCCAGTCACGACGTTGTAAA) (SEQ ID NO: 182). It can be seen thatthe Cas13b orthologs nos. 14, 15, 19, and 20 (from Porphyromonas gulae,Prevotella sp. P5-125, Porphyromonas gingivalis, and Porphyromonas sp.COT-0520H4946) are particularly active in mammalian cells.

FIG. 4 shows normalised data comparing the two most effective orthologs,14 and 15, with C2c2/Cas13a, using several luciferase-targeting guidesand a non-targeting control guide. It can be seen that the Cas13borthologs nos. 14 and 15 (from Porphyromonas gulae and Prevotella sp.P5-125) are consistently more active than Leptotrichia wadeii C2c2 inmammalian cells.

Example 3

Efficient and precise nucleic acid editing holds great promise fortreating genetic disease, particularly at the level of RNA, wheredisease-relevant transcripts can be rescued to yield functional proteinproducts. Type VI CRISPR-Cas systems contain the programmablesingle-effector RNA-guided RNases Cas13. Here, we profile the diversityof Type VI systems to engineer a Cas13 ortholog capable of robustknockdown and demonstrate RNA editing by using catalytically-inactiveCas13 (dCas13) to direct adenosine deaminase activity to transcripts inmammalian cells. By fusing the ADAR2 deaminase domain to dCas13 andengineering guide RNAs to create an optimal RNA duplex substrate, weachieve targeted editing of specific single adenosines to inosines(which is read out as guanosine during translation) with efficienciesroutinely ranging from 20-40% and up to 89%. This system, referred to asRNA Editing for Programmable A to I Replacement (REPAIR), can be furtherengineered to achieve high specificity. An engineered variant, REPAIRv2,displays greater than 170-fold increase in specificity while maintainingrobust on-target A to I editing. We use REPAIRv2 to edit full-lengthtranscripts containing known pathogenic mutations and create functionaltruncated versions suitable for packaging in adeno-associated viral(AAV) vectors. REPAIR presents a promising RNA editing platform withbroad applicability for research, therapeutics, and biotechnology.Precise nucleic acid editing technologies are valuable for studyingcellular function and as novel therapeutics. Although current editingtools, such as the Cas9 nuclease, can achieve programmable modificationof genomic loci, edits are often heterogenous due to insertions ordeletions or require a donor template for precise editing. Base editors,such as dCas9-APOBEC fusions, allow for editing without generating adouble stranded break, but may lack precision due to the nature ofcytidine deaminase activity, which edits any cytidine in a targetwindow. Furthermore, the requirement for a protospacer adjacent motif(PAM) limits the number of possible editing sites. Here, we describe thedevelopment of a precise and flexible RNA base editing tool using theRNA-guided RNA targeting Cas13 enzyme from type VI prokaryotic clusteredregularly interspaced short palindromic repeats (CRISPR) adaptive immunesystem.

Precise nucleic acid editing technologies are valuable for studyingcellular function and as novel therapeutics. Current editing tools,based on programmable nucleases such as the prokaryotic clusteredregularly interspaced short palindromic repeats (CRISPR)-associatednucleases Cas9 (1-4) or Cpf1 (5), have been widely adopted for mediatingtargeted DNA cleavage which in turn drives targeted gene disruptionthrough non-homologous end joining (NHEJ) or precise gene editingthrough template-dependent homology-directed repair (HDR)(6). NHEJutilizes host machineries that are active in both dividing andpost-mitotic cells and provides efficient gene disruption by generatinga mixture of insertion or deletion (indel) mutations that can lead toframe shifts in protein coding genes. HDR, in contrast, is mediated byhost machineries whose expression is largely limited to replicatingcells. As such, the development of gene-editing capabilities inpost-mitotic cells remains a major challenge. Recently, DNA baseeditors, such as the use of catalytically inactive Cas9 (dCas9) totarget cytidine deaminase activity to specific genome targets to effectcytosine to thymine conversions within a target window, allow forediting without generating a DNA double strand break and significantlyreduces the formation of indels (7, 8). However the targeting range ofDNA base editors is limited due to the requirement of Cas9 for aprotospacer adjacent motif (PAM) at the editing site (9). Here, wedescribe the development of a precise and flexible RNA base editingtechnology using the type VI CRISPR-associated RNA-guided RNaseCas13(10-13).

Cas13 enzymes have two Higher Eukaryotes and ProkaryotesNucleotide-binding (HEPN) endoRNase domains that mediate precise RNAcleavage (10, 11). Three Cas13 protein families have been identified todate: Cas13a (previously known as C2c2), Cas13b, and Cas13c (12, 13). Werecently reported Cas13a enzymes can be adapted as tools for nucleicacid detection (14) as well as mammalian and plant cell RNA knockdownand transcript tracking (15). The RNA-guided nature of Cas13 enzymesmakes them attractive tool for RNA binding and perturbationapplications.

The adenosine deaminase acting on RNA (ADAR) family of enzymes mediatesendogenous editing of transcripts via hydrolytic deamination ofadenosine to inosine, a nucleobase that is functionally equivalent toguanosine in translation and splicing (16). There are two functionalhuman ADAR orthologs, ADAR1 and ADAR2, which consist of N-terminaldouble stranded RNA-binding domains and a C-terminal catalyticdeamination domain. Endogenous target sites of ADAR1 and ADAR2 containsubstantial double stranded identity, and the catalytic domains requireduplexed regions for efficient editing in vitro and in vivo (17, 18).Although ADAR proteins have preferred motifs for editing that couldrestrict the potential flexibility of targeting, hyperactive mutants,such as ADAR(E488Q) (19), relax sequence constraints and improveadenosine to inosine editing rates. ADARs preferentially deaminateadenosines opposite cytidine bases in RNA duplexes (20), providing apromising opportunity for precise base editing. Although previousapproaches have engineered targeted ADAR fusions via RNA guides (21-24),the specificity of these approaches has not been reported and theirrespective targeting mechanisms rely on RNA-RNA hybridization withoutthe assistance of protein partners that may enhance target recognitionand stringency.

Here we assay the entire family of Cas13 enzymes for RNA knockdownactivity in mammalian cells and identify the Cas13b ortholog fromPrevotella sp. P5-125 (PspCas13b) as the most efficient and specific formammalian cell applications. We then fuse the ADAR2 deaminase domain(ADARDD) to catalytically inactive PspCas13b and demonstrate RNA editingfor programmable A to I (G) replacement (REPAIR) of reporter andendogenous transcripts as well as disease-relevant mutations. Lastly, weemploy a rational mutagenesis scheme to improve the specificity ofdCas13b-ADAR2DD fusions to generate REPAIRv2 with more than 170 foldincrease in specificity.

Design and Cloning of Bacterial Constructs

Mammalian codon optimized Cas13b constructs were cloned into thechloramphenicol resistant pACYC184 vector under control of the Lacpromoter. Two corresponding direct-repeat (DR) sequences separated byBsa1 restriction sites were then inserted downstream of Cas13b, undercontrol of the pJ23119 promoter. Last, oligos for targeting spacers werephosphorylated using T4 PNK (New England Biolabs), annealed and ligatedinto Bsa1 digested vectors using T7 ligase (Enzymatics) to generatetargeting Cas13b vectors.

Bacterial PFS Screens

Ampicillin resistance plasmids for PFS screens were cloned by insertingPCR products containing Cas13b targets with 2 5′ randomized nucleotidesand 4 3′ randomized nucleotides separated by a target site immediatelydownstream of the start codon of the ampicillin resistance gene b1ausing NEB Gibson Assembly (New England Biolabs). 100 ng ofampicillin-resistant target plasmids were then electroporated with65-100 ng chloramphenicol-resistant Cas13b bacterial targeting plasmidsinto Endura Electrocompetent Cells. Plasmids were added to cells,incubated 15 minutes on ice, electroporated using the manufacturer'sprotocol, and then 950 uL of recovery media was added to cells before aone hour outgrowth at 37C. The outgrowth was plated onto chloramphenicoland ampicillin double selection plates. Serial dilutions of theoutgrowth were used to estimate the cfu/ng DNA. 16 hours post plating,cells were scraped off plates and surviving plasmid DNA harvested usingthe Qiagen Plasmid Plus Maxi Kit (Qiagen). Surviving Cas13b targetsequences and their flanking regions were amplified by PCR and sequencedusing an Illumina NextSeq. To assess PFS preferences, the positionscontaining randomized nucleotides in the original library wereextracted, and sequences depleted relative to the vector only conditionthat were present in both bioreplicates were extracted using custompython scripts. The −log 2 of the ratio of PFS abundance in the Cas13bcondition compared to the vector only control was then used to calculatepreferred motifs. Specifically, all sequences having −log2(sample/vector) depletion ratios above a specific threshold were usedto generate weblogos of sequence motifs (weblogo.berkeley.edu). Thespecific depletion ratio values used to generate weblogos for eachCas13b ortholog are listed in Table 7.

Design and Cloning of Mammalian Constructs for RNA Interference

To generate vectors for testing Cas13 orthologs in mammalian cells,mammalian codon optimized Cas13a, Cas13b, and Cas13c genes were PCRamplified and golden-gate cloned into a mammalian expression vectorcontaining dual NLS sequences and a C-terminal msfGFP, under control ofthe EF1alpha promoter. For further optimization Cas13 orthologs weregolden gate cloned into destination vectors containing differentC-terminal localization tags under control of the EF1alpha promoter.

The dual luciferase reporter was cloned by PCR amplifying Gaussia andCypridinia luciferase coding DNA, the EF1alpha and CMV promoters andassembly using the NEB Gibson Assembly (New England Biolabs).

For expression of mammalian guide RNA for Cas13a, Cas13b, or Cas13corthologs, the corresponding direct repeat sequences were synthesizedwith golden-gate acceptor sites and cloned under U6 expression viarestriction digest cloning. Individual guides were then cloned into thecorresponding expression backbones for each ortholog by golden gatecloning.

Cloning of Pooled Mismatch Libraries for Cas13 Interference Specificity

Pooled mismatch library target sites were created by PCR. Oligoscontaining semi-degenerate target sequences in G-luciferase containing amixture of 94% of the correct base and 2% of each incorrect base at eachposition within the target were used as one primer, and an oligocorresponding to a non-targeted region of G-luciferase was used as thesecond primer in the PCR reaction. The mismatch library target was thencloned into the dual luciferase reporter in place of the wildtypeG-luciferase using NEB Gibson assembly (New England Biolabs).

Design and Cloning of Mammalian Constructs for RNA Editing

PspCas13b was made catalytically inactive (dPspCas13b) via two histidineto alanine mutations (H133A/H1058A) at the catalytic site of the HEPNdomains. The deaminase domains of human ADAR1 and ADAR2 were synthesizedand PCR amplified for gibson cloning into pcDNA-CMV vector backbones andwere fused to dPspCas13b at the C-terminus via GS or GSGGGGS (SEQ ID NO:183) linkers. For the experiment in which we tested different linkers wecloned the following additional linkers between dPspCas13b and ADAR2dd:GGGGSGGGGSGGGGS (SEQ ID NO: 184), EAAAK (SEQ ID NO: 185),GGSGGSGGSGGSGGSGGS (SEQ ID NO: 186), and SGSETPGTSESATPES (SEQ ID NO:187) (XTEN). Specificity mutants were generated by gibson cloning theappropriate mutants into the dPspCas13b-GSGGGGS (SEQ ID NO: 188)backbone.

The luciferase reporter vector for measuring RNA editing activity wasgenerated by creating a W85X mutation (TGG>TAG) in the luciferasereporter vector used for knockdown experiments. This reporter vectorexpresses functional Gluc as a normalization control, but a defectiveCluc due to the addition of a pretermination site. To test ADAR editingmotif preferences, we cloned every possible motif around the adenosineat codon 85 (XAX) of Cluc.

For testing PFS preference of REPAIR, we cloned a pooled plasmid librarycontaining a 6 basepair degenerate PFS sequence upstream of a targetregion and adenosine editing site. The library was synthesized as anultramer from Integrated DNA Technologies (IDT) and was made doublestranded via annealing a primer and Klenow fragment of DNA polymerase I(New England Biolabs) fill in of the sequence. This dsDNA fragmentcontaining the degenerate sequence was then gibson cloned into thedigested reporter vector and this was then isopropanol precipitated andpurified. The cloned library was then electroporated into Enduracompetent E. coli cells (Lucigen) and plated on 245 mm×245 mm squarebioassay plates (Nunc). After 16 hours, colonies were harvested andmidiprepped using endotoxin-free MACHEREY-NAGEL midiprep kits. Clonedlibraries were verified by next generation sequencing.

For cloning disease-relevant mutations for testing REPAIR activity, 34G>A mutations related to disease pathogenesis as defined in ClinVar wereselected and 200 bp regions surrounding these mutations were golden gatecloned between mScarlett and EGFP under a CMV promoter. Two additionalG>A mutations in AVPR2 and FANCC were selected for Gibson cloning thewhole gene sequence under expression of EF1alpha.

For expression of mammalian guide RNA for REPAIR, the PspCas13b directrepeat sequences were synthesized with golden-gate acceptor sites andcloned under U6 expression via restriction digest cloning. Individualguides were then cloned into this expression backbones by golden gatecloning.

Mammalian Cell Culture

Mammalian cell culture experiments were performed in the HEK293FT line(American Type Culture Collection (ATCC)), which was grown in Dulbecco'sModified Eagle Medium with high glucose, sodium pyruvate, and GlutaMAX(Thermo Fisher Scientific), additionally supplemented with 1×penicillin—streptomycin (Thermo Fisher Scientific) and 10% fetal bovineserum (VWR Seradigm). Cells were maintained at confluency below 80%.

Unless otherwise noted, all transfections were performed withLipofectamine 2000 (Thermo Fisher Scientific) in 96-well plates coatedwith poly-D-lysine (BD Biocoat). Cells were plated at approximately20,000 cells/well sixteen hours prior to transfection to ensure 90%confluency at the time of transfection. For each well on the plate,transfection plasmids were combined with Opti-MEM I Reduced Serum Medium(Thermo Fisher) to a total of 25 μl. Separately, 24.5 ul of Opti-MEM wascombined with 0.5 ul of Lipofectamine 2000. Plasmid and Lipofectaminesolutions were then combined and incubated for 5 minutes, after whichthey were pipetted onto cells.

RNA Knockdown Mammalian Cell Assays

To assess RNA targeting in mammalian cells with reporter constructs, 150ng of Cas13 construct was co-transfected with 300 ng of guide expressionplasmid and 12.5 ng of the knockdown reporter construct. 48 hourspost-transfection, media containing secreted luciferase was removed fromcells, diluted 1:5 in PBS, and measured for activity with BioLuxCypridinia and Biolux Gaussia luciferase assay kits (New EnglandBiolabs) on a plate reader (Biotek Synergy Neo2) with an injectionprotocol. All replicates performed are biological replicates.

For targeting of endogenous genes, 150 ng of Cas13 construct wasco-transfected with 300 ng of guide expression plasmid. 48 hourspost-transfection, cells were lysed and RNA was harvested and reversetranscribed using a previously described modification of the Cells-to-Ctkit (Thermo Fisher Scientific). cDNA expression was measured via qPCRusing TaqMan qPCR probes for the KRAS transcript (Thermo FisherScientific), GAPDH control probes (Thermo Fisher Scientific), and FastAdvanced Master Mix (Thermo Fisher Scientific). qPCR reactions were readout on a LightCycler 480 Instrument II (Roche), with four 5 ul technicalreplicates in 384-well format.

Evaluation of RNA Specificity Using Pooled Library of Mismatched Targets

The ability of Cas13 to interfere with the mismatched target library wastested using HEK293FT cells seeded in 6 well plates. ˜70% confluentcells were transfected using 2400 ng Cas13 vector, 4800 ng of guide and240 ng of mismatched target library. 48 hours post transfection, cellswere harvested and RNA extracted using the QIAshredder (Qiagen) and theQiagen RNeasy Mini Kit. lug of extracted RNA was reverse transcribedusing the qScript Flex cDNA synthesis kit (Quantabio) following themanufacturer's gene-specific priming protocol and a Gluc specific RTprimer. cDNA was then amplified and sequenced on an Illumina NextSeq.

The sequencing was analyzed by counting reads per sequence and depletionscores were calculated by determining the log 2(-read count ratio)value, where read count ratio is the ratio of read counts in thetargeting guide condition versus the non-targeting guide condition. Thisscore value represents the level of Cas13 activity on the sequence, withhigher values representing stronger depletion and thus higher Cas13cleavage activity. Separate distributions for the single mismatch anddouble mismatch sequences were determined and plotted as heatmaps with adepletion score for each mismatch identity. For double mismatchsequences the average of all possible double mismatches at a givenposition were plotted.

Transcriptome-Wide Profiling of Cas13 in Mammalian Cells by RNASequencing

For measurement of transcriptome-wide specificity, 150 ng of Cas13construct, 300 ng of guide expression plasmid and 15 ng of the knockdownreporter construct were co-transfected; for shRNA conditions, 300 ng ofshRNA targeting plasmid, 15 ng of the knockdown reporter construct, and150 ng of EF1-alpha driven mCherry (to balance reporter load) wereco-transfected. 48 hours after transfection, RNA was purified with theRNeasy Plus Mini kit (Qiagen), mRNA was selected for using NEBNextPoly(A) mRNA Magnetic Isolation Module (New England Biolabs) andprepared for sequencing with the NEBNext Ultra RNA Library Prep Kit forIllumina (New England Biolabs). RNA sequencing libraries were thensequenced on a NextSeq (Illumina).

To analyze transcriptome-wide sequencing data, reads were aligned RefSeqGRCh38 assembly using Bowtie and RSEM version 1.2.31 with defaultparameters. Transcript expression was quantified as log 2(TPM+1), geneswere filtered for log 2(TPM+1) >2.5 For selection of differentiallyexpressed genes, only genes with differential changes of >2 or <0.75were considered. Statistical significance of differential expression wasevaluated Student's T-test on three targeting replicates versusnon-targeting replicates, and filtered for a false discovery rate of<0.01% by Benjamini-Hochberg procedure.

ADAR RNA Editing in Mammalian Cells Transfections

To assess REPAIR activity in mammalian cells, we transfected 150 ng ofREPAIR vector, 300 ng of guide expression plasmid, and 40 ng of the RNAediting reporter. After 48 hours, RNA from cells were harvested andreverse transcribed using a method previously described with a genespecific reverse transcription primer. The extracted cDNA was thensubjected to two rounds of PCR to add Illumina adaptors and samplebarcodes using NEBNext High-Fidelity 2×PCR Master Mix. The library wasthen subjected to next generation sequencing on an Illumina NextSeq orMiSeq. RNA editing rates were then evaluated at all adenosine within thesequencing window.

In experiments where the luciferase reporter was targeted for RNAediting, we also harvested the media with secreted luciferase prior toRNA harvest. In this case, because the corrected Cluc might be at lowlevels, we did not dilute the media. We measured luciferase activitywith BioLux Cypridinia and Biolux Gaussia luciferase assay kits (NewEngland Biolabs) on a plate reader (Biotek Synergy Neo2) with aninjection protocol. All replicates performed are biological replicates.

PFS Binding Mammalian Screen

To determine the contribution of the PFS to editing efficiency, 625 ngof PFS target library, 4.7 ug of guide, and 2.35 ug of REPAIR wereco-transfected on HEK293FT cells plated in 225 cm2 flasks. Plasmids weremixed with 33 ul of PLUS reagent (Thermo Fisher Scientific), brought to533 ul with Opti-MEM, incubated for 5 minutes, combined with 30 ul ofLipofectamine 2000 and 500 ul of Opti-MEM, incubated for an additional 5minutes, and then pipetted onto cells. 48 hours post-transfection, RNAwas harvested with the RNeasy Plus Mini kit (Qiagen), reversetranscribed with qScript Flex (Quantabio) using a gene specific primer,and amplified with two rounds of PCR using NEBNext High-Fidelity 2×PCRMaster Mix (New England Biolabs) to add Illumina adaptors and samplebarcodes. The library was sequenced on an Illumina NextSeq, and RNAediting rates at the target adenosine were mapped to PFS identity. Toincrease coverage, the PFS was computationally collapsed to 4nucleotides. REPAIR editing rates were calculated for each PFS, averagedover biological replicates with non-targeting rates for thecorresponding PFS subtracted.

Whole-Transcriptome Sequencing to Evaluate ADAR Editing Specificity

For analyzing off-target RNA editing sites across the transcriptome, weharvested total RNA from cells 48 hours post transfection using theRNeasy Plus Miniprep kit (Qiagen). The mRNA fraction is then enrichedusing a NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) and thisRNA is then prepared for sequencing using NEBNext Ultra RNA Library PrepKit for Illumina (NEB). The libraries were then sequenced on an IlluminaNextSeq and loaded such that there was at least 5 million reads persample.

RNA Editing Analysis for Targeted and Transcriptome Wide Experiments

To analyze the transcriptome-wide RNA editing RNA sequencing data,sequence files were randomly downsampled to 5 million reads. An indexwas generated using the RefSeq GRCh38 assembly with Gluc and Clucsequences added and reads were aligned and quantified using Bowtie/RSEMversion 1.3.0. Alignment BAMs were then sorted and analyzed for RNAediting sites using REDitools [cite] with the following parameters: -t8-e-d-1-U [AG or TC]-p-u-m20-T6-0-W-v 1-n 0.0. Any significant editsfound in untransfected or EGFP-transfected conditions were considered tobe SNPs or artifacts of the transfection and filtered out from theanalysis of off-targets. Off-targets were considered significant if theFisher's exact test yielded a p-value less than 0.5 and that at least 2of 3 biological replicates identified the edit site.

For analyzing the predicted variant effects of each off-target, the listof off-target edit sites was analyzed using the variant annotationintegrator (https://genome.ucsc.edu/cgi-bin/hgVai) as part of the UCSCgenome browser suite of tools using the SIFT and PolyPhen-2 annotations.To declare whether the off-target genes are oncogenic, a database ofoncogenic annotations from the COSMIC catalogue of somatic mutations incancer (cancer. Sanger. ac.uk).

For analyzing whether the REPAIR constructs perturbed RNA levels, thetranscript per million (TPM) values output from the RSEM analysis wereused for expression counts and transformed to log-space by taking thelog 2(TPM+1). To find differentially regulated genes, a Student's t-testwas performed on three targeting guide replicates versus threenon-targeting guide replicates. The statistical analysis was onlyperformed on genes with log 2(TPM+1) values greater than 2.5 and geneswere only considered differentially regulated if they had a fold changegreater than 2 or less than 0.8. Genes were reported if they had a falsediscovery rate of less than 0.01.

Comprehensive Characterization of Cas13 Family Members in MammalianCells

We previously developed LwaCas13a for mammalian knockdown applications,but it required an msfGFP stabilization domain for efficient knockdownand, although the specificity was high, knockdown efficiencies were notconsistently below 50%(15). We sought to identify a more robustRNA-targeting CRISPR system by characterizing a genetically diverse setof Cas13 family members to assess their RNA knockdown activity inmammalian cells (FIG. 5A). We cloned 21 Cas13a, 15 Cas13b, and 7 Cas13cmammalian codon-optimized orthologs (Table 4) into an expression vectorwith N- and C-terminal nuclear export signal (NES) sequences and aC-terminal msfGFP to enhance protein stability. To assay interference inmammalian cells, we designed a dual reporter construct expressing theorthogonal Gaussia (Gluc) and Cypridinia (Cluc) luciferases underseparate promoters, which allows one luciferase to function as a measureof Cas13 interference activity and the other to serve as an internalcontrol. For each ortholog, we designed PFS-compatible guide RNAs, usingthe Cas13b PFS motifs derived from an ampicillin interference assay(FIG. 11; Table 5) and the 3′ H PFS from previous reports of Cas13aactivity (10).

We transfected HEK293FT cells with Cas13 expression, guide RNA andreporter plasmids and quantified levels of the targeted Gluc 48 hourslater. Testing two guide RNAs for each Cas13 ortholog revealed a rangeof activity levels, including five Cas13b orthologs with similar orincreased interference across both guide RNAs relative to the recentlycharacterized LwaCas13a (FIG. 5B). We selected these five Cas13borthologs, as well as the top two Cas13a orthologs for furtherengineering.

We next tested for Cas13-mediated knockdown of Gluc without msfGFP, inorder to select orthologs that do not require stabilization domains forrobust activity. We hypothesized that, in addition to msfGFP, Cas13activity could be affected by subcellular localization, as previouslyreported for optimization of LwaCas13a (15). Therefore, we tested theinterference activity of the seven selected Cas13 orthologs C-terminallyfused to one of six different localization tags without msfGFP. Usingthe luciferase reporter assay, we found that PspCas13b and PguCas13bC-terminally fused to the HIV Rev gene NES and RanCas13b C-terminallyfused to the MAPK NES had the highest levels of interference activity(FIG. 12A). To further distinguish activity levels of the top orthologs,we compared the three optimized Cas13b constructs to the optimalLwaCas13a-msfGFP fusion and shRNA for their ability to knockdown theKRAS transcript using position-matched guides (FIG. 12B). We observedthe highest levels interference for PspCas13b (average knockdown 62.9%)and thus selected this for further comparison to LwaCas13a.

To more rigorously define the activity level of PspCas13b and LwaCas13awe designed position matched guides tiling along both Gluc and Cluc andassayed their activity using our luciferase reporter assay. We tested 93and 20 position matched guides targeting Gluc and Cluc, respectively,and found that PspCas13b had consistently increased levels of knockdownrelative to LwaCas13a (average of 92.3% for PspCas13b vs. 40.1%knockdown for LwaCas13a) (FIG. 5C,D).

Specificity of Cas13 Mammalian Interference Activity

To characterize the interference specificities of PspCas13b andLwaCas13a we designed a plasmid library of luciferase targets containingsingle mismatches and double mismatches throughout the target sequenceand the three flanking 5′ and 3′ base pairs (FIG. 12C). We transfectedHEK293FT cells with either LwaCas13a or PspCas13b, a fixed guide RNAtargeting the unmodified target sequence, and the mismatched targetlibrary corresponding to the appropriate system. We then performedtargeted RNA sequencing of uncleaved transcripts to quantify depletionof mismatched target sequences. We found that LwaCas13a and PspCas13bhad a central region that was relatively intolerant to singlemismatches, extending from base pairs 12-26 for the PspCas13b target and13-24 for the LwaCas13a target (FIG. 12D). Double mismatches were evenless tolerated than single mutations, with little knockdown activityobserved over a larger window, extending from base pairs 12-29 forPspCas13b and 8-27 for LwaCas13a in their respective targets (FIG. 12E).Additionally, because there are mismatches included in the threenucleotides flanking the 5′ and 3′ ends of the target sequence, we couldassess PFS constraints on Cas13 knockdown activity. Sequencing showedthat almost all PFS combinations allowed robust knockdown, indicatingthat a PFS constraint for interference in mammalian cells likely doesnot exist for either enzyme tested. These results indicate that Cas13aand Cas13b display similar sequence constraints and sensitivitiesagainst mismatches.

We next characterized the interference specificity of PspCas13b andLwaCas13a across the mRNA fraction of the transcriptome. We performedtranscriptome-wide mRNA sequencing to detect significant differentiallyexpressed genes. LwaCas13a and PspCas13b demonstrated robust knockdownof Gluc (FIG. 5E,F) and were highly specific compared to aposition-matched shRNA, which showed hundreds of off-targets (FIG. 5G).

Cas13-ADAR Fusions Enable Targeted RNA Editing

Given that PspCas13b achieved consistent, robust, and specific knockdownof mRNA in mammalian cells, we envisioned that it could be adapted as anRNA binding platform to recruit the deaminase domain of ADARs (ADARDD)for programmable RNA editing. To engineer a PspCas13b lacking nucleaseactivity (dPspCas13b, referred to as dCas13b from here), we mutatedconserved catalytic residues in the HEPN domains and observed loss ofluciferase RNA knockdown activity (FIG. 13A). We hypothesized that adCas13b-ADAR_(DD) fusion could be recruited by a guide RNA to targetadenosines, with the hybridized RNA creating the required duplexsubstrate for ADAR activity (FIG. 6A). To enhance target adenosinedeamination rates we introduced two additional modifications to ourinitial RNA editing design: we introduced a mismatched cytidine oppositethe target adenosine, which has been previously reported to increasedeamination frequency, and fused dCas13b with the deaminase domains ofhuman ADAR1 or ADAR2 containing hyperactivating mutations to enhancecatalytic activity (ADAR1_(DD)(E1008Q)(25) or ADAR2_(DD)(E488Q)(/9)).

To test the activity of dCas13b-ADARDD we generated an RNA-editingreporter on Cluc by introducing a nonsense mutation (W85X (UGG->UAG)),which could functionally be repaired to the wildtype codon through A->Iediting (FIG. 6B) and then be detected as restoration of Clucluminescence. We evenly tiled guides with spacers 30, 50, 70 or 84nucleotides in length across the target adenosine to determine theoptimal guide placement and design (FIG. 6C). We found thatdCas13b-ADAR1DD required longer guides to repair the Cluc reporter,while dCas13b-ADAR2DD was functional with all guide lengths tested (FIG.6C). We also found that the hyperactive E488Q mutation improved editingefficiency, as luciferase restoration with the wildtype ADAR2_(DD) wasreduced (FIG. 13B). From this demonstration of activity, we chosedCas13b-ADAR2_(DD)(E488Q) for further characterization and designatedthis approach as RNA Editing for Programmable A to I Replacement version1 (REPAIRv1).

To validate that restoration of luciferase activity was due to bona fideediting events, we measured editing of Cluc transcripts subject toREPAIRv1 directly via reverse transcription and targeted next-generationsequencing. We tested 30- and 50-nt spacers around the target site andfound that both guide lengths resulted in the expected A to I edit, with50-nt spacers achieving higher editing percentages (FIG. 6D,E, FIG.13C). We also observed that 50-nt spacers had an increased propensityfor editing at non-targeted adenosines, likely due to increased regionsof duplex RNA (FIG. 6E, FIG. 13C).

We next targeted an endogenous gene, PPIB. We designed 50-nt spacerstiling PPIB and found that we could edit the PPIB transcript with up to28% editing efficiency (FIG. 13D). To test if REPAIR could be furtheroptimized, we modified the linker between dCas13b and ADAR2_(DD)(E488Q)(FIG. 13E, Table 6) and found that linker choice modestly affectedluciferase activity restoration.

Defining the Sequence Parameters for RNA Editing

Given that we could achieve precise RNA editing at a test site, wewanted to characterize the sequence constraints for programming thesystem against any RNA target in the transcriptome. Sequence constraintscould arise from dCas13b targeting limitations, such as the PFS, or fromADAR sequence preferences (26). To investigate PFS constraints onREPAIRv1, we designed a plasmid library carrying a series of fourrandomized nucleotides at the 5′ end of a target site on the Cluctranscript (FIG. 7A). We targeted the center adenosine within either aUAG or AAC motif and found that for both motifs, all PFSs demonstrateddetectable levels of RNA editing, with a majority of the PFSs havinggreater than 50% editing at the target site (FIG. 7B). Next, we soughtto determine if the ADAR2_(DD) in REPAIRv1 had any sequence constraintsimmediately flanking the targeted base, as has been reported previouslyfor ADAR2_(DD) (26). We tested every possible combination of 5′ and 3′flanking nucleotides directly surrounding the target adenosine (FIG.7C), and found that REPAIRv1 was capable of editing all motifs (FIG.7D). Lastly, we analyzed whether the identity of the base opposite thetarget A in the spacer sequence affected editing efficiency and foundthat an A-C mismatch had the highest luciferase restoration with A-G,A-U, and A-A having drastically reduced REPAIRv1 activity (FIG. 13F).

Correction of Disease-Relevant Human Mutations Using REPAIRv1

To demonstrate the broad applicability of the REPAIRv1 system for RNAediting in mammalian cells, we designed REPAIRv1 guides against twodisease relevant mutations: 878G>A (AVPR2 W293X) in X-linked Nephrogenicdiabetes insipidus and 1517G>A (FANCC W506X) in Fanconi anemia. Wetransfected expression constructs for cDNA of genes carrying thesemutations into HEK293FT cells and tested whether REPAIRv1 could correctthe mutations. Using guide RNAs containing 50-nt spacers, we were ableto achieve 35% correction of AVPR2 and 23% correction of FANCC (FIG.8A-D). We then tested the ability of REPAIRv1 to correct 34 differentdisease-relevant G>A mutations (Table 7) and found that we were able toachieve significant editing at 33 sites with up to 28% editingefficiency (FIG. 8E). The mutations we chose are only a fraction of thepathogenic G to A mutations (5,739) in the ClinVar database, which alsoincludes an additional 11,943 G to A variants (FIG. 8F and FIG. 14).Because there are no sequence constraints, REPAIRv1 is capable ofpotentially editing all these disease relevant mutations, especiallygiven that we observed significant editing regardless of the targetmotif (FIG. 7C and FIG. 8G).

Delivering the REPAIRv1 system to diseased cells is a prerequisite fortherapeutic use, and we therefore sought to design REPAIRv1 constructsthat could be packaged into therapeutically relevant viral vectors, suchas adeno-associated viral (AAV) vectors. AAV vectors have a packaginglimit of 4.7 kb, which cannot accommodate the large size ofdCas13b-ADARDD (4473 bp) along with promoter and expression regulatoryelements. To reduce the size, we tested a variety of N-terminal andC-terminal truncations of dCas13 fused to ADAR2_(DD)(E488Q) for RNAediting activity. We found that all C-terminal truncations tested werestill functional and able to restore luciferase signal (FIG. 15), andthe largest truncation, C-terminal 4984-1090 (total size of the fusionprotein 4,152 bp) was small enough to fit within the packaging limit ofAAV vectors.

Transcriptome-Wide Specificity of REPAIRv1

Although RNA knockdown with PspCas13b was highly specific, in ourluciferase tiling experiments, we observed off-target adenosine editingwithin the guide:target duplex (FIG. 6E). To see if this was awidespread phenomenon, we tiled an endogenous transcript, KRAS, andmeasured the degree of off-target editing near the target adenosine(FIG. 9A). We found that for KRAS, while the on-target editing rate was23%, there were many sites around the target site that also haddetectable A to G edits (FIG. 9B).

Because of the observed off-target editing within the guide:targetduplex, we evaluated all possible transcriptome off-targets byperforming RNA sequencing on all mRNAs. RNA sequencing revealed thatthere was a significant number A to G off-target events, with 1,732off-targets in the targeting condition and 925 off-targets in thenon-targeting condition, with 828 off-targets overlapping (FIG. 9C,D).Of all the editing sites across the transcriptome, the on-target editingsite had the highest editing rate, with 89% A to G conversion.

Given the high specificity of Cas13 targeting, we reasoned that theoff-targets may arise from ADAR. Two RNA-guided ADAR systems have beendescribed previously (FIG. 16A). The first utilizes a fusion of ADAR2DDto the small viral protein lambda N (λN), which binds to the BoxB-λ RNAhairpin (22). A guide RNA with double BoxB-λ hairpins guides ADAR2DD toedit sites encoded in the guide RNA (23). The second design utilizesfull length ADAR2 (ADAR2) and a guide RNA with a hairpin that the doublestrand RNA binding domains (dsRBDs) of ADAR2 recognize (21, 24). Weanalyzed the editing efficiency of these two systems compared toREPAIRv1 and found that the BoxB-ADAR2 and ADAR2 systems demonstrated63% and 36% editing rates, respectively, compared to the 89% editingrate achieved by REPAIRv1 (FIG. 16B-E). Additionally, the BoxB and ADAR2systems created 2018 and 174 observed off targets, respectively, in thetargeting guide conditions, compared to the 1,229 off targets in theREPAIRv1 targeting guide condition. Notably, all the conditions with thetwo ADAR2DD-based systems (REPAIRv1 and BoxB) showed a high percentageof overlap in their off-targets while the ADAR2 system had a largelydistinct set of off-targets (FIG. 16F). The overlap in off-targetsbetween the targeting and non-targeting conditions and between REPAIRv1and BoxB conditions suggest ADAR2_(DD) drove off-targets independent ofdCas13 targeting (FIG. 16F).

Improving Specificity of REPAIRv1 Through Rational Protein Engineering

To improve the specificity of REPAIR, we employed structure-guidedprotein engineering of ADAR2_(DD)(E488Q). Because of theguide-independent nature of off-targets, we hypothesized thatdestabilizing ADAR2_(DD)(E488Q)-RNA binding would selectively decreaseoff-target editing, but maintain on-target editing due to increasedlocal concentration from dCas13b tethering of ADAR2_(DD)(E488Q) to thetarget site. We mutagenized ADAR2_(DD)(E488Q) residues previouslydetermined to contact the duplex region of the target RNA (FIG. 10A)(18) on the ADAR2_(DD)(E488Q) background. To assess efficiency andspecificity, we tested 17 single mutants with both targeting andnon-targeting guides, under the assumption that background luciferaserestoration in the non-targeting condition detected would be indicativeof broader off-target activity. We found that mutations at the selectedresidues had significant effects on the luciferase activity fortargeting and non-targeting guides (FIG. 10A,B, FIG. 17A). A majority ofmutants either significantly improved the luciferase activity for thetargeting guide or increased the ratio of targeting to non-targetingguide activity, which we termed the specificity score (FIG. 10A,B). Weselected a subset of these mutants (FIG. 10B) for transcriptome-widespecificity profiling by next generation sequencing. As expected,off-targets measured from transcriptome-wide sequencing correlated withour specificity score (FIG. 17B) for mutants. We found that with theexception of ADAR2_(DD)(E488Q/R455E), all sequenced REPAIRv1 mutantscould effectively edit the reporter transcript (FIG. 10C), with manymutants showing reduction in the number of off-targets (FIGS. 17C and18). We further explored the surrounding motifs of off-targets forspecificity mutants, and found that REPAIRv1 and most of the engineeredmutants exhibited a strong 3′ G preference for their edits, in agreementwith the characterized ADAR2 motif (FIG. 19A) (26). We selected themutant ADAR2_(DD)(E488Q/T375G) for future experiments, as it had thehighest percent editing of the four mutants with the lowest numbers oftranscriptome-wide off targets and termed it REPAIRv2. Compared toREPAIRv1, REPAIRv2 exhibited increased specificity, with a reductionfrom 1732 to 10 transcriptome off-targets (FIG. 10D). In the regionsurrounding the targeted adenosine in Cluc, REPAIRv2 had reducedoff-target editing, visible in sequencing traces (FIG. 10E). In motifsderived from next-generation sequencing, REPAIRv1 presented a strongpreference towards 3′ G, but showed off-targeting edits for all motifs(FIG. 19B); by contrast, REPAIRv2 only edited the strongest off-targetmotifs (FIG. 19C). The distribution of edits on transcripts was heavilyskewed, with highly-edited genes having over 60 edits (FIG. 20A,B),whereas REPAIRv2 only edited one transcript (EEF1A1) multiple times(FIG. 20D-F). REPAIRv1 off-target edits were predicted to result innumerous variants, including 1000 missense mutations (FIG. 20C) with 93oncogenic events (FIG. 20D). In contrast, REPAIRv2 only had 6 missensemutations (FIG. 20E), none of which had oncogenic consequences (FIG.20F). This reduction in predicted off-target effects distinguishesREPAIRv2 from other RNA editing approaches.

We targeted REPAIRv2 to endogenous genes to test if thespecificity-enhancing mutations reduced nearby edits in targettranscripts while maintaining high-efficiency on-target editing. Forguides targeting either KRAS or PPIB, we found that REPAIRv2 had nodetectable off-target edits, unlike REPAIRv1, and could effectively editthe on-target adenosine at 27.1% and 13%, respectively (FIG. 10F,G).This specificity extended to additional target sites, including regionsthat demonstrate high-levels of background in non-targeting conditionsfor REPAIRv1, such as other KRAS or PPIB target sites (FIG. 21).Overall, REPAIRv2 eliminated off-targets in duplexed regions around theedited adenosine and showed dramatically enhanced transcriptome-widespecificity.

CONCLUSION

We have shown here that the RNA-guided RNA-targeting type VI-B effectorCas13b is capable of highly efficient and specific RNA knockdown,providing the basis for improved tools for interrogating essential genesand non-coding RNA as well as controlling cellular processes at thetranscriptomic level. Catalytically inactive Cas13b (dCas13b) retainsprogrammable RNA binding capability, which we leveraged here by fusingdCas13b to the adenosine deaminase ADAR2 to achieve precise A to Iedits, a system we term REPAIRv1 (RNA Editing for Programmable A to IReplacement version 1). Further engineering of the system producedREPAIRv2, a method with comparable or increased activity relative tocurrent editing platforms with dramatically improved specificity.

Although Cas13b exhibits high fidelity, our initial results withdCas13b-ADAR2_(DD) fusions revealed thousands of off-targets. To addressthis, we employed a rational mutagenesis strategy to vary the ADAR2_(DD)residues that contact the RNA duplex, identifying a variant,ADAR2_(DD)(E488Q/T375G), capable of precise, efficient, and highlyspecific editing when fused to dCas13b. Editing efficiency with thisvariant was comparable to or better than that achieved with twocurrently available systems, BoxB-ADAR_(DD) or ADAR2 editing. Moreover,the REPAIRv2 system created only 10 observable off-targets in the wholetranscriptome, at least an order of magnitude better than bothalternative editing technologies.

The REPAIR system offers many advantages compared to other nucleic acidediting tools. First, the exact target site can be encoded in the guideby placing a cytidine within the guide extension across from the desiredadenosine to create a favorable A-C mismatch ideal for ADAR editingactivity. Second, Cas13 has no targeting sequence constraints, such as aPFS or PAM, and no motif preference surrounding the target adenosine,allowing any adenosine in the transcriptome to be potentially targetedwith the REPAIR system. We do note, however, that DNA base editors cantarget either the sense or anti-sense strand, while the REPAIR system islimited to transcribed sequences, thereby constraining the total numberof possible editing sites we could target. However, due to the moreflexible nature of targeting with REPAIR, this system can effect moreedits within ClinVar (FIG. 8C) than Cas9-DNA base editors. Third, theREPAIR system directly deaminates target adenosines to inosines and doesnot rely on endogenous repair pathways, such as base-excision ormismatch repair, to generate desired editing outcomes. Thus, REPAIRshould be possible in non-dividing cells that cannot support other formsof editing. Fourth, RNA editing can be transient, allowing the potentialfor temporal control over editing outcomes. This property will likely beuseful for treating diseases caused by temporary changes in cell state,such as local inflammation.

The REPAIR system provides multiple opportunities for additionalengineering. Cas13b possesses pre-crRNA processing activity (13),allowing for multiplex editing of multiple variants, which alone mightnot alter disease risk, but together might have additive effects anddisease-modifying potential. Extension of our rational design approach,such as combining promising mutations, could further increase thespecificity and efficiency of the system, while unbiased screeningapproaches could identify additional residues for improving REPAIRactivity and specificity.

Currently, the base conversions achievable by REPAIR are limited togenerating inosine from adenosine; additional fusions of dCas13 withother catalytic RNA editing domains, such as APOBEC, could enablecytidine to uridine editing. Additionally, mutagenesis of ADAR couldrelax the substrate preference to target cytidine, allowing for theenhanced specificity conferred by the duplexed RNA substrate requirementto be exploited by C->U editors. Adenosine to inosine editing on DNAsubstrates may also be possible with catalytically inactiveDNA-targeting CRISPR effectors, such as dCas9 or dCpf1, either throughformation of DNA-RNA heteroduplex targets (27) or mutagenesis of theADAR domain.

REPAIR could be applied towards a range of therapeutic indications whereA to I (A to G) editing can reverse or slow disease progression (FIG.22). First, expression of REPAIR for targeting causal, Mendelian G to Amutations in disease-relevant tissues could be used to revertdeleterious mutations and treat disease. For example, stable REPAIRexpression via AAV in brain tissue could be used to correct the GRIN2Amissense mutation c.2191G>A (Asp731Asn) that causes focal epilepsy (28)or the APP missense mutation c.2149G>A (Val1717Ile) causing early-onsetAlzheimer's disease (29). Second, REPAIR could be used to treat diseaseby modifying the function of proteins involved in disease-related signaltransduction. For instance, REPAIR editing would allow the re-coding ofsome serine, threonine and tyrosine residues that are the targets ofkinases (FIG. 22). Phosphorylation of these residues in disease-relevantproteins affects disease progression for many disorders includingAlzheimer's disease and multiple neurodegenerative conditions (30).Third, REPAIR could be used to change the sequence of expressed,risk-modifying G to A variants to pre-emptively decrease the chance ofentering a disease state for patients. The most intriguing case are the‘protective’ risk-modifying alleles, which dramatically decrease thechance of entering a disease state, and in some cases, confer additionalhealth benefits. For instance, REPAIR could be used to functionallymimic A to G alleles of PCSK9 and IFIH1 that protect againstcardiovascular disease and psoriatic arthritis (31), respectively. Last,REPAIR can be used to therapeutically modify splice acceptor and donorsites for exon modulation therapies. REPAIR can change AU to IU or AA toAI, the functional equivalent of the consensus 5′ splice donor or 3′splice acceptor sites respectively, creating new splice junctions.Additionally, REPAIR editing can mutate the consensus 3′ splice acceptorsite from AG->IG to promote skipping of the adjacent downstream exon, atherapeutic strategy that has received significant interest for thetreatment of DMD. Modulation of splice sites could have broadapplications in diseases where anti-sense oligos have had some success,such as for modulation of SMN2 splicing for treatment of spinal muscularatrophy (32).

We have demonstrated the use of the PspCas13b enzyme as both an RNAknockdown and RNA editing tool. The dCas13b platform for programmableRNA binding has many applications, including live transcript imaging,splicing modification, targeted localization of transcripts, pull downof RNA-binding proteins, and epitranscriptomic modifications. Here, weused dCas13 to create REPAIR, adding to the existing suite of nucleicacid editing technologies. REPAIR provides a new approach for treatinggenetic disease or mimicking protective alleles, and establishes RNAediting as a useful tool for modifying genetic function.

TABLE 4 Cas13 Orthologs used in this study Cas13 Cas13 ID abbreviationHost Organism Protein Accession Cas13a1 LshCas13a Leptotrichia shahiiWP_018451595.1 Cas13a2 LwaCas13a Leptotrichia wadei (Lw2) WP_021746774.1Cas13a3 LseCas13a Listeria seeligeri WP_012985477.1 Cas13a4 LbmCas13aLachnospiraceae bacterium WP_044921188.1 MA2020 Cas13a5 LbnCas13aLachnospiraceae bacterium WP_022785443.1 NK4A179 Cas13a6 CamCas13a[Clostridium] aminophilum DSM WP_031473346.1 10710 Cas13a7 CgaCas13aCarnobacterium gallinarum DSM WP_034560163.1 4847 Cas13a8 Cga2Cas13aCarnobacterium gallinarum DSM WP_034563842.1 4847 Cas13a9 Pprcas13aPaludibacter propionicigenes WP_013443710.1 WB4 Cas13a10 LweCas13aListeria weihenstephanensis FSL WP_036059185.1 R9-0317 Cas13a11LbfCas13a Listeriaceae bacterium FSL M6- WP_036091002.1 0635 Cas13a12Lwa2Cas13a Leptotrichia wadei F0279 WP_021746774.1 Cas13a13 RcsCas13aRhodobacter capsulatus SB 1003 WP_013067728.1 Cas13a14 RcrCas13aRhodobacter capsulatus R121 WP_023911507.1 Cas13a15 RcdCas13aRhodobacter capsulatus DE442 WP_023911507.1 Cas13a16 LbuCas13aLeptotrichia buccalis C-1013-b WP_015770004.1 Cas13a17 HheCas13aHerbinix hemicellulosilytica CRZ35554.1 Cas13a18 EreCas13a [Eubacterium]rectale WP_055061018.1 Cas13a19 EbaCas13a Eubacteriaceae bacteriumWP_090127496.1 CHKCI004 Cas13a20 BmaCas13a Blautia sp. Marseille-P2398WP_062808098.1 Cas13a21 LspCas13a Leptotrichia sp. oral taxon 879WP_021744063.1 str. F0557 Cas13b1 BzoCas13b Bergeyella zoohelcumWP_002664492 Cas13b2 PinCas13b Prevotella intermedia WP_036860899Cas13b3 PbuCas13b Prevotella buccae WP_004343973 Cas13b4 AspCas13bAlistipes sp. ZOR0009 WP_047447901 Cas13b5 PsmCas13b Prevotella sp.MA2016 WP_036929175 Cas13b6 RanCas13b Riemerella anatipestiferWP_004919755 Cas13b7 PauCas13b Prevotella aurantiaca WP_025000926Cas13b8 PsaCas13b Prevotella saccharolytica WP_051522484 Cas13b9Pin2Cas13b Prevotella intermedia WP_061868553 Cas13b10 CcaCas13bCapnocytophaga canimorsus WP_013997271 Cas13b11 PguCas13b Porphyromonasgulae WP_039434803 Cas13b12 PspCas13b Prevotella sp. P5-125 WP_044065294Cas13b13 FbrCas13b Flavobacterium branchiophilum WP_014084666 Cas13b14PgiCas13b Porphyromonas gingivalis WP_053444417 Cas13b15 Pin3Cas13bPrevotella intermedia WP_050955369 Cas13c1 FnsCas13c Fusobacteriumnecrophorum WP_005959231.1 subsp. funduliforme ATCC 51357 contig00003Cas13c2 FndCas13c Fusobacterium necrophorum DJ- WP_035906563.1 2contig0065, whole genome shotgun sequence Cas13c3 FnbCas13cFusobacterium necrophorum WP_035935671.1 BFTR-1 contig0068 Cas13c4FnfCas13c Fusobacterium necrophorum EHO19081.1 subsp. funduliforme1_1_36S cont1.14 Cas13c5 FpeCas13c Fusobacterium perfoetens ATCCWP_027128616.1 29250 T364DRAFT_scaffold00009.9_C Cas13c6 FulCas13cFusobacterium ulcerans ATCC WP_040490876.1 49185 cont2.38 Cas13c7AspCas13c Anaerosalibacter sp. ND1 WP_042678931.1 genome assemblyAnaerosalibacter massiliensis ND1

TABLE 5 PFS cutoffs in bacterial screens −Log₂ depletion score used toCas13b ortholog Key generate PFS motif Bergeyella zoohelcum 1 2Prevotella intermedia locus 1 2 1 Prevotella buccae 3 3 Alistipes sp.ZOR0009 4 1 Prevotella sp. MA2016 5 2 Riemerella anatipestifer 6 4Prevotella aurantiaca 7 1 Prevotella saccharolytica 8 0 Prevotellaintermedia locus 2 9 0 Capnocytophaga canimorsus 10 3 Porphyromonasgulae 11 4 Prevotella sp. P5-125 12 2.1 Flavobacterium 13 1branchiophilum Porphyromonas gingivalis 14 3 Prevotella intermedia locus2 15 4

TABLE 6 dCas13b-ADAR linker sequences used in thisstudy for RNA editing in mammalian cells. Figure linker 6CGSGGGGS (SEQ ID NO: 189) 6E GS 13B GSGGGGS (SEQ ID NO: 190) 13C GS 13DGS 13E: GS GS 13E: GSGGGGS GSGGGGS (SEQ ID NO: 190) 13E: (GGGS)3GGGGSGGGGSGGGGS (SEQ ID NO: 191) 13E: Rigid EAAAK (SEQ ID NO: 192)13E: (GGS)6 GGSGGSGGSGGSGGSGGS (SEQ ID NO: 193) 13E: XTENSGSETPGTSESATPES (SEQ ID NO: 194) 7B GS 13F GS 7C GS 8B GS 8D GS 8E GS7A: Δ984-1090, GS Δ1026-1090, Δ1053-1090 7A: Δ1-125,GSGGGGS (SEQ ID NO: 190) Δ1-88, Δ1-72 9B GS 9C GS 9D GS 16A GS 16C GS16D GS 17D GS 10A GS 18A GS 10B GS 18B GS 18C GS 19A GS 19B GS 10C GS10D GS 10E GS 1OF GS 22A GS 22A GS

TABLE 7 Disease information for disease-relevant mutationsFull length candidates Gene Disease NM_000054.4(AVPR2):c.878G>A AVPR2Nephrogenic diabetes insipidus, (p.Trp293Ter) X-linkedNM_000136.2(FANCC):c.1517G>A FANCC Fanconi anemia, (p.Trp506Ter)complementation group C Additional simulated candiates Candidate GeneDisease NM_000206.2(IL2RG):c.710G>A IL2RG X-linked severe combined(p.Trp237Ter) immunodeficiency NM_000132.3(F8):c.3144G>A F8Hereditary factor VIII (p.Trp1048Ter) deficiency diseaseNM_000527.4(LDLR):c.1449G>A LDLR Familial hypercholesterolemia(p.Trp483Ter) NM_0000712(CBS):c.162G>A CBS Homocystinuria due to CBS(p.Trp54Ter) deficiency NM_000518.4(HBB):c.114G>A HBB beta{circumflexover ( )}0{circumflex over ( )} Thalassemia|beta (p.Trp38Ter)Thalassemia NM_000035.3(ALDOB):c.888G>A ALDOB Hereditary fructosuria(p.Trp296Ter) NM_004006.2(DMD):c.3747G>A DMD Duchenne muscular dystrophy(p.Trp1249Ter) NM_005359.5(SMAD4):c.906G>A SMAD4Juvenile polyposis syndrome (p.Trp302Ter) NM_000059.3(BRCA2):c.582G>ABRCA2 Familial cancer of breast|Breast- (p.Trp194Ter)ovarian cancer, familial 2 NM_000833.4(GRIN2A):c.3813G>A GRIN2AEpilepsy, focal, with speech (p.Trp1271Ter) disorder and with or withoutmental retardation NM_002977.3(SCN9A):c.2691G>A SCN9AIndifference to pain, congenital, (p.Trp897Ter) autosomal recessiveNM_007375.3(TARDBP):c.943G>A TARDBP Amyotrophic lateral sclerosis(p.Ala315Thr) type 10 NM_000492.3(CFTR):c.3846G>A CFTRCystic fibrosis|Hereditary (p.Trp1282Ter) pancreatitis|notprovided|ataluren response- Efficacy NM_130838.1(UBE3A):c.2304G>A UBE3AAngelman syndrome (p.Trp768Ter) NM_000543.4(SMPD1):c.168G>A SMPDINiemann-Pick disease, type A (p.Trp56Ter) NM_206933.2(USH2A):c.9390G>AUSH2A Usher syndrome, type 2A (p.Trp3130Ter) NM_130799.2(MEN1):c.1269G>AMEN1 Hereditary cancer-predisposing (p.Trp423Ter) syndromeNM_177965.3(C8orf37):c.555G>A C8orf37 Retinitis pigmentosa 64(p.Trp185Ter) NM_000249.3(MLH1):c.1998G>A MLH1 Lynch syndrome(p.Trp666Ter) NM_000548.4(TSC2):c.2108G>A TSC2Tuberous sclerosis 2|Tuberous (p.Trp703Ter) sclerosis syndromeNM_000267.3(NF1):c.7044G>A NF1 Neurofibromatosis, type 1 (p.Trp2348Ter)NM_000179.2(MSH6):c.3020G>A MSH6 Lynch syndrome (p.Trp1007Ter)NM_000344.3(SMN1):c.305G>A SMN1 Spinal muscular atrophy, type(p.Trp102Ter) II|Kugelberg-Welander disease NM_024577.3(SH3TC2):c.920G>ASH3TC2 Charcot-Marie-Tooth disease, (p.Trp307Ter) type 4CNM_001369.2(DNAH5):c.8465G>A DNAH5 Primary ciliary dyskinesia(p.Trp2822Ter) NM_004992.3(MECP2):c.311G>A MECP2 Rett syndrome(p.Trp104Ter) NM_032119.3(ADGRV1):c.7406G>A ADGRV1Usher syndrome, type 2C (p.Trp2469Ter) NM_017651,4(AHI1):c.2174G>A AHI1Joubert syndrome 3 (p.Trp725Ter) NM_004562.2(PRKN):c.1358G>A PRKNParkinson disease 2 (p.Trp453Ter) NM_000090.3(COL3A1):c.3833G>A COL3A1Ehlers-Danlos syndrome, type 4 (p.Trp1278Ter)NM_007294.3(BRCA1):c.5511G>A BRCA1 Familial cancer of breast|Breast-(p.Trp1837Ter) ovarian cancer, familial 1 NM_000256.3(MYBPC3):c.3293G>AMYBPC3 Primary familial hypertrophic (p.Trp1098Ter) cardiomyopathyNM_000038.5(APC):c.1262G>A APC Familial adenomatous polyposis(p.Trp421Ter) 1 NM_001204.6(BMPR2):c.893G>A BMPR2 Primary pulmonary(P.W298*) hypertension

TABLE 8 Key plasmids used in this study Plasmid name Description pAB0006CMV-Cluciferase-polyA EF1a-G-luciferase-poly A pAB0040CMV-Cluciferase(STOP85)-polyA EF1a-G-luciferase- polyA pAB0048pCDNA-ADAR2-N-terminal B12-HIV NES pAB0050 pAB0001-A02 (crRNA backbone)pAB0051 pAB0001-B06 (crRNA backbone) pAB0052 pAB0001-B11 (crRNAbackbone) pAB0053 pAB0001-B12 (crRNA backbone) pAB0014.B6EF1a-BsiWI-Cas13b6-NES-mapk pAB0013.B11 EF1a-BsiWI-Cas13b11-NES-HIVpAB0013.B12 EF1a-BsiWI-Cas13b12-NES-HIV pAB0051 pAB0001-B06 (crRNAbackbone) pAB0052 pAB0001-B11 (crRNA backbone) pAB0053 pAB0001-B12(crRNA backbone) pAB0079 pCDNA-ADAR1hu-EQmutant-N-terminal destinationvector pAB0085 pCDNA-ADAR2 (E488Q)hu-EQmutant-N-terminal destinationvector pAB0095 EF1a-BsiWI-Cas13-B12-NES-HIV, with double H HEPN mutantpAB0114 pCDNA-wtADAR2hu-EQmutant-N-terminal destination vector pAB0120Luciferase ADAR guide optimal (guide 24 from wC0054) pAB0122 pAB0001-B12NT guide for ADAR experiments pAB0151 pCDNA-ADAR2hu-EQmutant-N-terminaldestination vector C-term delta 984-1090 pAB0180 T375G specificitymutant pAB0181 T375G Cas13b C-term delta 984-1090

TABLE 9 Guide/shRNA sequences used in this studyfor knockdown in mammalian cells Interference First Name Spacer sequenceMechanism Notes FIG. Bacterial GCCAGCUUUCCGGGCAUUGG Cas13b Used for allPFS guide CUUCCAUC (SEQ ID NO: orthologs 195) Cas13a-GCCAGCTTTCCGGGCATTGG Cas13a Used for all FIG. 5B GlucCTTCCATC (SEQ ID NO: Cas13a guide 1 196) orthologs Cas13a-ACCCAGGAATCTCAGGAATG Cas13a Used for all FIG. 5B GlucTCGACGAT (SEQ ID NO: Cas13a guide 2 197) orthologs Cas13a-AGGGTTTTCCCAGTCACGAC Cas13a Used for all FIG. 5B non-GTTGTAAA (SEQ ID NO: Cas13a targeting 198) orthologs guide (LacZ)Cas13b- GGGCATTGGCTTCCATCTCTT Cas13b Used for FIG. 5B GlucTGAGCACCT (SEQ ID NO: orthologs 1-3, guide 1.1 199) 6, 7, 10, 11,12, 14, 15 Cas13b- GUGCAGCCAGCUUUCCGGGC Cas13b Used for FIG. 5B GlucAUUGGCUUCC (SEQ ID ortholog 4 guide 1.2 NO: 200) Cas13b-GCAGCCAGCUUUCCGGGCAU Cas13b Used for FIG. 5B Gluc UGGCUUCCAU (SEQ IDortholog 5 guide 1.3 NO: 201) Cas13b- GGCUUCCAUCUCUUUGAGCA Cas13bUsed for FIG. 5B Gluc CCUCCAGCGG (SEQ ID ortholog 8, 9 guide 1.4NO: 202) Cas13b- GGAAUGUCGACGAUCGCCUC Cas13b Used for FIG. 5B GlucGCCUAUGCCG (SEQ ID ortholog 13 guide 1.5 NO: 203) Cas13b-GAAUGUCGACGAUCGCCUCG Cas13b Used for FIG. 5B Gluc CCUAUGCCGC (SEQ IDorthologs 1-3, guide 2.1 NO: 204) 6, 7, 10, 11, 14, 15 Cas13b-GACCUGUGCGAUGAACUGCU Cas13b Used for FIG. 5B Gluc CCAUGGGCUC (SEQ IDortholog 12 guide 2.2 NO: 205) Cas13b- GUGUGGCAGCGUCCUGGGA Cas13bUsed for FIG. 5B Gluc UGAACUUCUUC (SEQ ID ortholog 4 guide 2.2 NO: 206)Cas13b- GUGGCAGCGUCCUGGGAUG Cas13b Used for FIG. 5B GlucAACUUCUUCAU (SEQ ID ortholog 5 guide 2.3 NO: 207) Cas13b-GCUUCUUGCCGGGCAACUUC Cas13b Used for FIG. 5B Gluc CCGCGGUCAG (SEQ IDortholog 8, 9 guide 2.4 NO: 208) Cas13b- GCAGGGUUUUCCCAGUCACG Cas13bUsed for FIG. 5B Gluc ACGUUGUAAAA (SEQ ID ortholog 13 guide 2.6 NO: 209)Cas13b- GCAGGGUUUUCCCAGUCACG Cas13b Used for all FIG. 5B nonACGUUGUAAAA (SEQ ID orthologs targeting NO: 210) guide Cas13a-ACCCAGGAAUCUCAGGAAUG Cas13a FIG. 5E Gluc UCGACGAU (SEQ ID NO: guide-211) RNASeq shRNA- CAGCTTTCCGGGCATTGGCTT shRNA FIG. 5F Gluc(SEQ ID NO: 212) guide Cas13b- CCGCUGGAGGUGCUCAAAGA Cas13b FIG. 5F GlucGAUGGAAGCC (SEQ ID guide- NO: 213) RNASeq Cas13a- GCCAGCTTTCCGGGCATTGGCas13a FIG. 12A Gluc- CTTCCATC (SEQ ID NO: guide-1 214) Cas13a-ACCCAGGAATCTCAGGAATG Cas13a FIG. 12A Gluc- TCGACGAT (SEQ ID NO: guide-2215) Cas13b- GGGCATTGGCTTCCATCTCTT Cas13b FIG. 12A Gluc-opt-TGAGCACCT (SEQ ID NO: guide-1 216) Cas13b- GAAUGUCGACGAUCGCCUCG Cas13bFIG. 12A Gluc-opt- CCUAUGCCGC (SEQ ID guide-2 NO: 217) Cas13aCAAGGCACTCTTGCCTACGC Cas13a FIG. 12B KRAS CACCAGCT (SEQ ID NO: guide 1218) Cas13a TCATATTCGTCCACAAAATG Cas13a FIG. 12B KRASATTCTGAA (SEQ ID NO: guide 2 219) Cas13a ATTATTTATGGCAAATACAC Cas13aFIG. 12B KRAS AAAGAAAG (SEQ ID NO: guide 3 220) Cas13aGAATATCTTCAAATGATTTAG Cas13a FIG. 12B KRAS TATTATT (SEQ ID NO: guide 4221) Cas13a ACCATAGGTACATCTTCAGA Cas13a FIG. 12B KRASGTCCTTAA (SEQ ID NO: guide 5 222) Cas13b GTCAAGGCACTCTTGCCTAC Cas13bFIG. 12B KRAS GCCACCAGCT (SEQ ID guide 1 NO: 223) Cas13bGATCATATTCGTCCACAAAA Cas13b FIG. 12B KRAS TGATTCTGAA (SEQ ID guide 2NO: 224) Cas13b GTATTATTTATGGCAAATAC Cas13b FIG. 12B KRASACAAAGAAAG (SEQ ID guide 3 NO: 225) Cas13b GTGAATATCTTCAAATGATT Cas13bFIG. 12B KRAS TAGTATTATT (SEQ ID guide 4 NO: 226) Cas13bGGACCATAGGTACATCTTCA Cas13b FIG. 12B KRAS GAGTCCTTAA (SEQ ID guide 5NO: 227) shRNA aagagtgccttgacgataca shRNA FIG. 12B KRASgcCTCGAGgctgtatcgtca guide 1 aggcactctt (SEQ ID NO: 228) shRNAaatcattttgtggacgaata shRNA FIG. 12B KRAS tCTCGAGatattcgtccaca guide 2aaatgatt (SEQ ID NO: 229) shRNA aaataatactaaatcatttg shRNA FIG. 12B KRASaCTCGAGtcaaatgatttag guide 3 tattattt (SEQ ID NO: 230) shRNAaataatactaaatcatttga shRNA FIG. 12B KRAS aCTCGAGttcaaatgattta guide 4gtattatt (SEQ ID NO: 231) shRNA aaggactctgaagatgtacc shRNA FIG. 12B KRAStCTCGAGaggtacatcttca guide 5 gagtcctt (SEQ ID NO: 232)

TABLE 10 Guide sequences used for Gluc knockdown First NameSpacer sequence Position Notes FIG. Gluc GAGATCAGGGCAAACAG 2Note that the Cas13a spacers 5C tiling AACTTTGACTCCC (SEQare truncated by two guide 1 ID NO: 233) nucleotides at the 5′ end GlucGGATGCAGATCAGGGCA 7 Note that the Cas13a spacers 5C tilingAACAGAACTTTGA (SEQ are truncated by two guide 2 ID NO: 234)nucleotides at the 5′ end Gluc GCACAGCGATGCAGATCA 13Note that the Cas13a spacers 5C tiling GGGCAAACAGAA (SEQ IDare truncated by two guide 3 NO: 235) nucleotides at the 5′ end GlucGCTCGGCCACAGCGATGC 19 Note that the Cas13a spacers 5C tilingAGATCAGGGCAA (SEQ ID are truncated by two guide 4 NO: 236)nucleotides at the 5′ end Gluc GGGGCTTGGCCTCGGCCA 28Note that the Cas13a spacers 5C tiling CAGCGATGCAGA (SEQ IDare truncated by two guide 5 NO: 237) nucleotides at the 5′ end GlucGTGGGCTTGGCCTCGGCC 29 Note that the Cas13a spacers 5C tilingACAGCGATGCAG (SEQ ID are truncated by two guide 6 NO: 238nucleotides at the 5′ end Gluc GTCTCGGTGGGCTTGGCC 35Note that the Cas13a spacers 5C tiling TCGGCCACAGCG (SEQ IDare truncated by two guide 7 NO: 239) nucleotides at the 5′ end GlucGTTCGTTGTTCTCGGTGG 43 Note that the Cas13a spacers 5C tilingGCTTGGCCTCGG (SEQ ID are truncated by two guide 8 NO: 240)nucleotides at the 5′ end Gluc GGAAGTCTTCGTTGTTCT 49Note that the Cas13a spacers 5C tiling CGGTGGGCTTGG (SEQ IDare truncated by two guide 9 NO: 241) nucleotides at the 5′ end GlucGATGTTGAAGTCTTCGTT 54 Note that the Cas13a spacers 5C tilingGTTCTCGGTGGG (SEQ ID are truncated by two guide 10 NO: 242)nucleotides at the 5′ end Gluc GCGGCCACGATGTTGAAG 62Note that the Cas13a spacers 5C tiling TCTTCGTTGTTC (SEQ IDare truncated by two guide 11 NO: 243) nucleotides at the 5′ end GlucGTGGCCACGGCCACGATG 68 Note that the Cas13a spacers 5C tilingTTGAAGTCTTCG (SEQ ID are truncated by two guide 12 NO: 244)nucleotides at the 5′ end Gluc GGTTGCTGGCCACGGCCA 73Note that the Cas13a spacers 5C tiling CGATGTTGAAGT (SEQ IDare truncated by two guide 13 NO: 245) nucleotides at the 5′ end GlucGTCGCGAAGTTGCTGGCC 80 Note that the Cas13a spacers 5C tilingACGGCCACGATG (SEQ ID are truncated by two guide 14 NO: 246)nucleotides at the 5′ end Gluc GCCGTGGTCGCGAAGTTG 86Note that the Cas13a spacers 5C tiling CTGGCCACGGCC (SEQ IDare truncated by two guide 15 NO: 247) nucleotides at the 5′ end GlucGCGAGATCCGTGGTCGCG 92 Note that the Cas13a spacers 5C tilingAAGTTGCTGGCC (SEQ ID are truncated by two guide 16 NO: 248)nucleotides at the 5′ end Gluc GCAGCATCGAGATCCGTG 98Note that the Cas13a spacers 5C tiling GTCGCGAAGTTG (SEQ IDare truncated by two guide 17 NO: 249) nucleotides at the 5′ end GlucGGGTCAGCATCGAGATCC 101 Note that the Cas13a spacers 5C tilingGTGGTCGCGAAG (SEQ ID are truncated by two guide 18 NO: 250)nucleotides at the 5′ end Gluc GCTTCCCGCGGTCAGCAT 109Note that the Cas13a spacers 5C tiling CGAGATCCGTGG (SEQ IDare truncated by two guide 19 NO: 251) nucleotides at the 5′ end GlucGGGGCAACTTCCCGCGGT 115 Note that the Cas13a spacers 5C tilingCAGCATCGAGAT (SEQ ID are truncated by two guide 20 NO: 252)nucleotides at the 5′ end Gluc GTCTTGCCGGGCAACTTC 122Note that the Cas13a spacers 5C tiling CCGCGGTCAGCA (SEQ IDare truncated by two guide 21 NO: 253) nucleotides at the 5′ end GlucGGCAGCTTCTTGCCGGGC 128 Note that the Cas13a spacers 5C tilingAACTTCCCGCGG (SEQ ID are truncated by two guide 22 NO: 254)nucleotides at the 5′ end Gluc GCCAGCGGCAGCTTCTTG 134Note that the Cas13a spacers 5C tiling CCGGGCAACTTC (SEQ IDare truncated by two guide 23 NO: 255) nucleotides at the 5′ end GlucGCACCTCCAGCGGCAGCT 139 Note that the Cas13a spacers 5C tilingTCTTGCCGGGCA (SEQ ID are truncated by two guide 24 NO: 256)nucleotides at the 5′ end Gluc GCTTTGAGCACCTCCAGC 146Note that the Cas13a spacers 5C tiling GGCAGCTTCTTG (SEQ IDare truncated by two guide 25 NO: 257) nucleotides at the 5′ end GlucGCATCTCTTTGAGCACCT 151 Note that the Cas13a spacers 5C tilingCCAGCGGCAGCT (SEQ ID are truncated by two guide 26 NO: 258)nucleotides at the 5′ end Gluc GTCCATCTCTTTGAGCAC 153Note that the Cas13a spacers 5C tiling CTCCAGCGGCAG (SEQ IDare truncated by two guide 27 NO: 259) nucleotides at the 5′ end GlucGGGCATTGGCTTCCATCT 163 Note that the Cas13a spacers 5C tilingCTTTGAGCACCT (SEQ ID are truncated by two guide 28 NO: 260)nucleotides at the 5′ end Gluc GTCCGGGCATTGGCTTCC 167Note that the Cas13a spacers 5C tiling ATCTCTTTGAGC (SEQ IDare truncated by two guide 29 NO: 261) nucleotides at the 5′ end GlucGGCCAGCTTTCCGGGCAT 175 Note that the Cas13a spacers 5C tilingTGGCTTCCATCT (SEQ ID are truncated by two guide 30 NO: 262)nucleotides at the 5′ end Gluc GGGTGCAGCCAGCTTTCC 181Note that the Cas13a spacers 5C tiling GGGCATTGGCTT (SEQ IDare truncated by two guide 31 NO: 263) nucleotides at the 5′ end GlucGAGCCCCTGGTGCAGCCA 188 Note that the Cas13a spacers 5C tilingGCTTTCCGGGCA (SEQ ID are truncated by two guide 32 NO: 264)nucleotides at the 5′ end Gluc GATCAGACAGCCCCTGGT 195Note that the Cas13a spacers 5C tiling GCAGCCAGCTTT (SEQ IDare truncated by two guide 33 NO: 265) nucleotides at the 5′ end GlucGGCAGATCAGACAGCCCC 199 Note that the Cas13a spacers 5C tilingTGGTGCAGCCAG (SEQ ID are truncated by two guide 34 NO: 266)nucleotides at the 5′ end Gluc GACAGGCAGATCAGACA 203Note that the Cas13a spacers 5C tiling GCCCCTGGTGCAG (SEQare truncated by two guide 35 ID NO: 267) nucleotides at the 5′ end GlucGTGATGTGGGACAGGCA 212 Note that the Cas13a spacers 5C tilingGATCAGACAGCCC (SEQ are truncated by two guide 36 ID NO: 268)nucleotides at the 5′ end Gluc GACTTGATGTGGGACAGG 215Note that the Cas13a spacers 5C tiling CAGATCAGACAG (SEQ IDare truncated by two guide 37 NO: 269) nucleotides at the 5′ end GlucGGGGCGTGCACTTGATGT 223 Note that the Cas13a spacers 5C tilingGGGACAGGCAGA (SEQ ID are truncated by two guide 38 NO: 270)nucleotides at the 5′ end Gluc GCTTCATCTTGGGCGTGC 232Note that the Cas13a spacers 5C tiling ACTTGATGTGGG (SEQ IDare truncated by two guide 39 NO: 271) nucleotides at the 5′ end GlucGTGAACTTCTTCATCTTG 239 Note that the Cas13a spacers 5C tilingGGCGTGCACTTG (SEQ ID are truncated by two guide 40 NO: 272)nucleotides at the 5′ end Gluc GGGATGAACTTCTTCATC 242Note that the Cas13a spacers 5C tiling TTGGGCGTGCAC (SEQ IDare truncated by two guide 41 NO: 273) nucleotides at the 5′ end GlucGTGGGATGAACTTCTTCA 244 Note that the Cas13a spacers 5C tilingTCTTGGGCGTGC (SEQ ID are truncated by two guide 42 NO: 274)nucleotides at the 5′ end Gluc GGGCAGCGTCCTGGGATG 254Note that the Cas13a spacers 5C tiling AACTTCTTCATC (SEQ IDare truncated by two guide 43 NO: 275) nucleotides at the 5′ end GlucGGGTGTGGCAGCGTCCTG 259 Note that the Cas13a spacers 5C tilingGGATGAACTTCT (SEQ ID are truncated by two guide 44 NO: 276)nucleotides at the 5′ end Gluc GTTCGTAGGTGTGGCAGC 265Note that the Cas13a spacers 5C tiling GTCCTGGGATGA (SEQ IDare truncated by two guide 45 NO: 277) nucleotides at the 5′ end GlucGCGCCTTCGTAGGTGTGG 269 Note that the Cas13a spacers 5C tilingCAGCGTCCTGGG (SEQ ID are truncated by two guide 46 NO: 278)nucleotides at the 5′ end Gluc GTCTTTGTCGCCTTCGTA 276Note that the Cas13a spacers 5C tiling GGTGTGGCAGCG (SEQ IDare truncated by two guide 47 NO: 279) nucleotides at the 5′ end GlucGCTTTGTCGCCTTCGTAG 275 Note that the Cas13a spacers 5C tilingGTGTGGCAGCGT (SEQ ID are truncated by two guide 48 NO: 280)nucleotides at the 5′ end Gluc GTGCCGCCCTGTGCGGAC 293Note that the Cas13a spacers 5C tiling TCTTTGTCGCCT (SEQ IDare truncated by two guide 49 NO: 281) nucleotides at the 5′ end GlucGTATGCCGCCCTGTGCGG 295 Note that the Cas13a spacers 5C tilingACTCTTTGTCGC (SEQ ID are truncated by two guide 50 NO: 282)nucleotides at the 5′ end Gluc GCCTCGCCTATGCCGCCC 302Note that the Cas13a spacers 5C tiling TGTGCGGACTCT (SEQ IDare truncated by two guide 51 NO: 283) nucleotides at the 5′ end GlucGGATCGCCTCGCCTATGC 307 Note that the Cas13a spacers 5C tilingCGCCCTGTGCGG (SEQ ID are truncated by two guide 52 NO: 284)nucleotides at the 5′ end Gluc GATGTCGACGATCGCCTC 315Note that the Cas13a spacers 5C tiling GCCTATGCCGCC (SEQ IDare truncated by two guide 53 NO: 285) nucleotides at the 5′ end GlucGCAGGAATGTCGACGATC 320 Note that the Cas13a spacers 5C tilingGCCTCGCCTATG (SEQ ID are truncated by two guide 54 NO: 286)nucleotides at the 5′ end Gluc GAATCTCAGGAATGTCGA 325Note that the Cas13a spacers 5C tiling CGATCGCCTCGC (SEQ IDare truncated by two guide 55 NO: 287) nucleotides at the 5′ end GlucGCCCAGGAATCTCAGGAA 331 Note that the Cas13a spacers 5C tilingTGTCGACGATCG (SEQ ID are truncated by two guide 56 NO: 288)nucleotides at the 5′ end Gluc GCCTTGAACCCAGGAATC 338Note that the Cas13a spacers 5C tiling TCAGGAATGTCG (SEQ IDare truncated by two guide 57 NO: 289) nucleotides at the 5′ end GlucGCCAAGTCCTTGAACCCA 344 Note that the Cas13a spacers 5C tilingGGAATCTCAGGA (SEQ ID are truncated by two guide 58 NO: 290)nucleotides at the 5′ end Gluc GTGGGCTCCAAGTCCTTG 350Note that the Cas13a spacers 5C tiling AACCCAGGAATC (SEQ IDare truncated by two guide 59 NO: 291) nucleotides at the 5′ end GlucGCCATGGGCTCCAAGTCC 353 Note that the Cas13a spacers 5C tilingTTGAACCCAGGA (SEQ ID are truncated by two guide 60 NO: 292)nucleotides at the 5′ end Gluc GGAACTGCTCCATGGGCT 361Note that the Cas13a spacers 5C tiling CCAAGTCCTTGA (SEQ IDare truncated by two guide 61 NO: 293) nucleotides at the 5′ end GlucGTGCGATGAACTGCTCCA 367 Note that the Cas13a spacers 5C tilingTGGGCTCCAAGT (SEQ ID are truncated by two guide 62 NO: 294)nucleotides at the 5′ end Gluc GGACCTGTGCGATGAACT 373Note that the Cas13a spacers 5C tiling GCTCCATGGGCT (SEQ IDare truncated by two guide 63 NO: 295) nucleotides at the 5′ end GlucGACAGATCGACCTGTGCG 380 Note that the Cas13a spacers 5C tilingATGAACTGCTCC (SEQ ID are truncated by two guide 64 NO: 296)nucleotides at the 5′ end Gluc GACACACAGATCGACCTG 384Note that the Cas13a spacers 5C tiling TGCGATGAACTG (SEQ IDare truncated by two guide 65 NO: 297) nucleotides at the 5′ end GlucGTGCAGTCCACACACAGA 392 Note that the Cas13a spacers 5C tilingTCGACCTGTGCG (SEQ ID are truncated by two guide 66 NO: 298)nucleotides at the 5′ end Gluc GCCAGTTGTGCAGTCCAC 399Note that the Cas13a spacers 5C tiling ACACAGATCGAC (SEQ IDare truncated by two guide 67 NO: 299) nucleotides at the 5′ end GlucGGGCAGCCAGTTGTGCAG 404 Note that the Cas13a spacers 5C tilingTCCACACACAGA (SEQ ID are truncated by two guide 68 NO: 300)nucleotides at the 5′ end Gluc GTTTGAGGCAGCCAGTTG 409Note that the Cas13a spacers 5C tiling TGCAGTCCACAC (SEQ IDare truncated by two guide 69 NO: 301) nucleotides at the 5′ end GlucGAAGCCCTTTGAGGCAGC 415 Note that the Cas13a spacers 5C tilingCAGTTGTGCAGT (SEQ ID are truncated by two guide 70 NO: 302)nucleotides at the 5′ end Gluc GCACGTTGGCAAGCCCTT 424Note that the Cas13a spacers 5C tiling TGAGGCAGCCAG (SEQ IDare truncated by two guide 71 NO: 303) nucleotides at the 5′ end GlucGACTGCACGTTGGCAAGC 428 Note that the Cas13a spacers 5C tilingCCTTTGAGGCAG (SEQ ID are truncated by two guide 72 NO: 304)nucleotides at the 5′ end Gluc GGGTCAGAACACTGCACG 437Note that the Cas13a spacers 5C tiling TTGGCAAGCCCT (SEQ IDare truncated by two guide 73 NO: 305) nucleotides at the 5′ end GlucGCAGGTCAGAACACTGCA 439 Note that the Cas13a spacers 5C tilingCGTTGGCAAGCC (SEQ ID are truncated by two guide 74 NO: 306)nucleotides at the 5′ end Gluc GAGCAGGTCAGAACACT 441Note that the Cas13a spacers 5C tiling GCACGTTGGCAAG (SEQare truncated by two guide 75 ID NO: 307) nucleotides at the 5′ end GlucGGCCACTTCTTGAGCAGG 452 Note that the Cas13a spacers 5C tilingTCAGAACACTGC (SEQ ID are truncated by two guide 76 NO: 308)nucleotides at the 5′ end Gluc GCGGCAGCCACTTCTTGA 457Note that the Cas13a spacers 5C tiling GCAGGTCAGAAC (SEQ IDare truncated by two guide 77 NO: 309) nucleotides at the 5′ end GlucGTGCGGCAGCCACTTCTT 459 Note that the Cas13a spacers 5C tilingGAGCAGGTCAGA (SEQ ID are truncated by two guide 78 NO: 310)nucleotides at the 5′ end Gluc GAGCGTTGCGGCAGCCAC 464Note that the Cas13a spacers 5C tiling TTCTTGAGCAGG (SEQ IDare truncated by two guide 79 NO: 311) nucleotides at the 5′ end GlucGAAAGGTCGCACAGCGTT 475 Note that the Cas13a spacers 5C tilingGCGGCAGCCACT (SEQ ID are truncated by two guide 80 NO: 312)nucleotides at the 5′ end Gluc GCTGGCAAAGGTCGCACA 480Note that the Cas13a spacers 5C tiling GCGTTGCGGCAG (SEQ IDare truncated by two guide 81 NO: 313) nucleotides at the 5′ end GlucGGGCAAAGGTCGCACAG 478 Note that the Cas13a spacers 5C tilingCGTTGCGGCAGCC (SEQ are truncated by two guide 82 ID NO: 314)nucleotides at the 5′ end Gluc GTGGATCTTGCTGGCAAA 489Note that the Cas13a spacers 5C tiling GGTCGCACAGCG (SEQ IDare truncated by two guide 83 NO: 315) nucleotides at the 5′ end GlucGCACCTGGCCCTGGATCT 499 Note that the Cas13a spacers 5C tilingTGCTGGCAAAGG (SEQ ID are truncated by two guide 84 NO: 316)nucleotides at the 5′ end Gluc GTGGCCCTGGATCTTGCT 495Note that the Cas13a spacers 5C tiling GGCAAAGGTCGC (SEQ IDare truncated by two guide 85 NO: 317) nucleotides at the 5′ end GlucGTGATCTTGTCCACCTGG 509 Note that the Cas13a spacers 5C tilingCCCTGGATCTTG (SEQ ID are truncated by two guide 86 NO: 318)nucleotides at the 5′ end Gluc GCCCCTTGATCTTGTCCA 514Note that the Cas13a spacers 5C tiling CCTGGCCCTGGA (SEQ IDare truncated by two guide 87 NO: 319) nucleotides at the 5′ end GlucGCCCTTGATCTTGTCCAC 513 Note that the Cas13a spacers 5C tilingCTGGCCCTGGAT (SEQ ID are truncated by two guide 88 NO: 320)nucleotides at the 5′ end Gluc GCCTTGATCTTGTCCACC 512Note that the Cas13a spacers 5C tiling TGGCCCTGGATC (SEQ IDare truncated by two guide 89 NO: 321) nucleotides at the 5′ end GlucGGCAAAGGTCGCACAGC 477 Note that the Cas13a spacers 5C tilingGTTGCGGCAGCCA (SEQ are truncated by two guide 90 ID NO: 322)nucleotides at the 5′ end Gluc GCAAAGGTCGCACAGCGT 476Note that the Cas13a spacers 5C tiling TGCGGCAGCCAC (SEQ IDare truncated by two guide 91 NO: 323) nucleotides at the 5′ end GlucGAAGGTCGCACAGCGTTG 474 Note that the Cas13a spacers 5C tilingCGGCAGCCACTT (SEQ ID are truncated by two guide 92 NO: 324)nucleotides at the 5′ end Gluc GAGGTCGCACAGCGTTGC 473Note that the Cas13a spacers 5C tiling GGCAGCCACTTC (SEQ IDare truncated by two guide 93 NO: 325) nucleotides at the 5′ end Non-GGTAATGCCTGGCTTGTC N/A Note that the Cas13a spacers 5C targetingGACGCATAGTCTG (SEQ are truncated by two guide 1 ID NO: 326)nucleotides at the 5′ end Non- GGGAACCTTGGCCGTTAT N/ANote that the Cas13a spacers 5C targeting AAAGTCTGACCAG (SEQare truncated by two guide 2 ID NO: 327) nucleotides at the 5′ end Non-GGAGGGTGAGAATTTAG N/A Note that the Cas13a spacers 5C targetingAACCAAGATTGTTG (SEQ are truncated by two guide 3 ID NO: 328)nucleotides at the 5′ end

TABLE 11 Guide sequences used for Cluc knockdown First NameSpacer sequence Position Notes FIG. Cluc GAGTCCTGGCAATGAACA 32Note that the Cas13a spacers 5D tiling GTGGCGCAGTAG (SEQ IDare truncated by two guide 1 NO: 329) nucleotides at the 5′ end ClucGGGTGCCACAGCTGCTAT 118 Note that the Cas13a spacers 5D tilingCAATACATTCTC (SEQ ID are truncated by two guide 2 NO: 330)nucleotides at the 5′ end Cluc GTTACATACTGACACATT 197Note that the Cas13a spacers 5D tiling CGGCAACATGTT (SEQ IDare truncated by two guide 3 NO: 331) nucleotides at the 5′ end ClucGTATGTACCAGGTTCCTG 276 Note that the Cas13a spacers 5D tilingGAACTGGAATCT (SEQ ID are truncated by two guide 4 NO: 332)nucleotides at the 5′ end Cluc GCCTTGGTTCCATCCAGG 350Note that the Cas13a spacers 5D tiling TTCTCCAGGGTG (SEQ IDare truncated by two guide 5 NO: 333) nucleotides at the 5′ end ClucGCAGTGATGGGATTCTCA 431 Note that the Cas13a spacers 5D tilingGTAGCTTGAGCG (SEQ ID are truncated by two guide 6 NO: 334)nucleotides at the 5′ end Cluc GAGCCTGGCATCTCAACA 512Note that the Cas13a spacers 5D tiling ACAGCGATGGTG (SEQ IDare truncated by two guide 7 NO: 335) nucleotides at the 5′ end ClucGTGTCTGGGGCGATTCTT 593 Note that the Cas13a spacers 5D tilingACAGATCTTCCT (SEQ ID are truncated by two guide 8 NO: 336)nucleotides at the 5′ end Cluc GCTGGATCTGAAGTGAAG 671Note that the Cas13a spacers 5D tiling TCTGTATCTTCC (SEQ IDare truncated by two guide 9 NO: 337) nucleotides at the 5′ end ClucGGCAACGTCATCAGGATT 747 Note that the Cas13a spacers 5D tilingTCCATAGAGTGG (SEQ ID are truncated by two guide 10 NO: 338)nucleotides at the 5′ end Cluc GAGGCGCAGGAGATGGT 830Note that the Cas13a spacers 5D tiling GTAGTAGTAGAAG (SEQare truncated by two guide 11 ID NO: 339) nucleotides at the 5′ end ClucGAGGGACCCTGGAATTGG 986 Note that the Cas13a spacers 5D tilingTATCTTGCTTTG (SEQ ID are truncated by two guide 13 NO: 340)nucleotides at the 5′ end Cluc GGTAAGAGTCAACATTCC 1066Note that the Cas13a spacers 5D tiling TGTGTGAAACCT (SEQ IDare truncated by two guide 14 NO: 341) nucleotides at the 5′ end ClucGACCAGAATCTGTTTTCC 1143 Note that the Cas13a spacers 5D tilingATCAACAATGAG (SEQ ID are truncated by two guide 15 NO: 342)nucleotides at the 5′ end Cluc GATGGCTGTAGTCAGTAT 1227Note that the Cas13a spacers 5D tiling GTCACCATCTTG (SEQ IDare truncated by two guide 16 NO: 343) nucleotides at the 5′ end ClucGTACCATCGAATGGATCT 1304 Note that the Cas13a spacers 5D tilingCTAATATGTACG (SEQ ID are truncated by two guide 17 NO: 344)nucleotides at the 5′ end Cluc GAGATCACAGGCTCCTTC 1380Note that the Cas13a spacers 5D tiling AGCATCAAAAGA (SEQ IDare truncated by two guide 18 NO: 345) nucleotides at the 5′ end ClucGCTTTGACCGGCGAAGAG 1461 Note that the Cas13a spacers 5D tilingACTATTGCAGAG (SEQ ID are truncated by two guide 19 NO: 346)nucleotides at the 5′ end Cluc GCCCCTCAGGCAATACTC 1539Note that the Cas13a spacers 5D tiling GTACATGCATCG (SEQ IDare truncated by two guide 20 NO: 347) nucleotides at the 5′ end ClucGCTGGTACTTCTAGGGTG 1619 Note that the Cas13a spacers 5D tilingTCTCCATGCTTT (SEQ ID are truncated by two guide 21 NO: 348)nucleotides at the 5′ end Non- GGTAATGCCTGGCTTGTC N/ANote that the Cas13a spacers 5D targeting GACGCATAGTCTG (SEQare truncated by two guide 1 ID NO: 349) nucleotides at the 5′ end Non-GGGAACCTTGGCCGTTAT N/A Note that the Cas13a spacers 5D targetingAAAGTCTGACCAG (SEQ are truncated by two guide 2 ID NO: 350)nucleotides at the 5′ end Non- GGAGGGTGAGAATTTAG N/ANote that the Cas13a spacers 5D targeting AACCAAGATTGTTG (SEQare truncated by two guide 3 ID NO: 351) nucleotides at the 5′ end

TABLE 12Guide sequences used in this study for RNA editing in mammalian cells.Mismatched base flips are capitalized First Name Spacer sequence NotesFIG. Tiling 30 nt 30 gCatcctgeggcctctactctgcattcaattHas a 5′ G for U6 expression 6C mismatch distance (SEQ ID NO: 352)Tiling 30 nt 28 gacCatcctgeggcctctactctgcattcaaHas a 5′ G for U6 expression 6C mismatch distance (SEQ ID NO: 353)Tiling 30 nt 26 gaaacCatcctgcggcctctactctgcattcHas a 5′ G for U6 expression 6C mismatch distance (SEQ ID NO: 354)Tiling 30 nt 24 gctaaacCatcctgcggcctctactctgcatHas a 5′ G for U6 expression 6C mismatch distance (SEQ ID NO: 355)Tiling 30 nt 22 gttctaaacCatcctgcggcctctactctgcHas a 5′ G for U6 expression 6C mismatch distance (SEQ ID NO: 356)Tiling 30 nt 20 gtgttctaaacCatcctgcggcctctactctHas a 5′ G for U6 expression 6C mismatch distance (SEQ ID NO: 357)Tiling 30 nt 18 gaatgttctaaacCatcctgcggcctctactHas a 5′ G for U6 expression 6C mismatch distance (SEQ ID NO: 358)Tiling 30 nt 16 gagaatgttctaaacCatcctgcggcctctaHas a 5′ G for U6 expression 6C mismatch distance (SEQ ID NO: 359)Tiling 30 nt 14 gatagaatgttctaaacCatcctgcggcctcHas a 5′ G for U6 expression 6C mismatch distance (SEQ ID NO: 360)Tiling 30 nt 12 gccatagaatgttctaaacCatcctgcggccHas a 5′ G for U6 expression 6C mismatch distance (SEQ ID NO: 361)Tiling 30 nt 10 gttccatagaatgttctaaacCatcctgcggHas a 5′ G for U6 expression 6C mismatch distance (SEQ ID NO: 362)Tiling 30 nt 8 gctaccatagaatgttctaaacCatcctgcHas a 5′ G for U6 expression 6C mismatch distance (SEQ ID NO: 363)Tiling 30 nt 6 gctctaccatagaatgttctaaacCatcctHas a 5′ G for U6 expression 6C mismatch distance (SEQ ID NO: 364)Tiling 30 nt 4 gatctctaccatagaatgttctaaacCatcHas a 5′ G for U6 expression 6C mismatch distance (SEQ ID NO: 365)Tiling 30 nt 2 ggaatctctaccatagaatgttctaaacCaHas a 5′ G for U6 expression 6C mismatch distance (SEQ ID NO: 366)Tiling 50 nt 50 gCatcctgcggcctctactctgcattcaattHas a 5′ G for U6 expression 6C mismatch distanceacatactgacacattcggca(SEQ ID NO: 367) Tiling 50 nt 48gacCatcctgcggcctctactctgcattcaa Has a 5′ G for U6 expression 6Cmismatch distance ttacatactgacacattcgg(SEQ ID NO: 368) Tiling 50 nt 46gaaacCatcctgcggcctctactctgcattc Has a 5′ G for U6 expression 6Cmismatch distance aattacatactgacacattc(SEQ ID NO: 369) Tiling 50 nt 44gctaaacCatcctgcggcctctactctgcat Has a 5′ G for U6 expression 6Cmismatch distance tcaattacatactgacacat(SEQ ID NO: 370) Tiling 50 nt 42gttctaaacCatcctgcggcctctactctgc Has a 5′ G for U6 expression 6Cmismatch distance attcaattacatactgacac(SEQ ID NO: 371) Tiling 50 nt 40gtgttctaaacCatcctgcggcctctactct Has a 5′ G for U6 expression 6Cmismatch distance gcattcaattacatactgac(SEQ ID NO: 372) Tiling 50 nt 38gaatgttctaaacCatcctgcggcctctact Has a 5′ G for U6 expression 6Cmismatch distance ctgcattcaattacatactg(SEQ ID NO: 373) Tiling 50 nt 36gagaatgttctaaacCatcctgcggcctcta Has a 5′ G for U6 expression 6Cmismatch distance ctctgcattcaattacatac(SEQ ID NO: 374) Tiling 50 nt 34gatagaatgttctaaacCatcctgcggcctc Has a 5′ G for U6 expression 6Cmismatch distance tactctgcattcaattacat(SEQ ID NO: 375) Tiling 50 nt 32gccatagaatgttctaaacCatcctgcggcc Has a 5′ G for U6 expression 6Cmismatch distance tctactctgcattcaattac(SEQ ID NO: 376) Tiling 50 nt 30gttccatagaatgttctaaacCatcctgcgg Has a 5′ G for U6 expression 6Cmismatch distance cctctactctgcattcaatt(SEQ ID NO: 377) Tiling 50 nt 28gctaccatagaatgttctaaacCatcctgcg Has a 5′ G for U6 expression 6Cmismatch distance gcctctactctgcattcaa(SEQ ID NO: 378) Tiling 50 nt 26gctctaccatagaatgttctaaacCatcctg Has a 5′ G for U6 expression 6Cmismatch distance cggcctctactctgcattc(SEQ ID NO: 379) Tiling 50 nt 24gatctctaccatagaatgttctaaacCatcc Has a 5′ G for U6 expression 6Cmismatch distance tgcggcctctactctgcat(SEQ ID NO: 380) Tiling 50 nt 22ggaatctctaccatagaatgttctaaacCat Has a 5′ G for U6 expression 6Cmismatch distance cctgcggcctctactctgc(SEQ ID NO: 381) Tiling 50 nt 20gtggaatctctaccatagaatgttctaaacC Has a 5′ G for U6 expression 6Cmismatch distance atcctgcggcctctactct(SEQ ID NO: 382) Tiling 50 nt 18gactggaatctctaccatagaatgttctaaa Has a 5′ G for U6 expression 6Cmismatch distance cCatcctgcggcctctact(SEQ ID NO: 383) Tiling 50 nt 16ggaactggaatctctaccatagaatgttcta Has a 5′ G for U6 expression 6Cmismatch distance aacCatcctgcggcctcta(SEQ ID NO: 384) Tiling 50 nt 14gtggaactggaatctctttccatagaatgtt Has a 5′ G for U6 expression 6Cmismatch distance ctaaacCatcctgcggcctc(SEQ ID NO; 385) Tiling 50 nt 12gcctggaactggaatctctttccatagaatg Has a 5′ G for U6 expression 6Cmismatch distance ttctaaacCatcctgcggcc(SEQ ID NO: 386) Tiling 50 nt 10gttcctggaactggaatctctaccatagaat Has a 5′ G for U6 expression 6Cmismatch distance gttctaaacCatcctgcgg(SEQ ID NO: 387) Tiling 50 nt 8gggttcctggaactggaatctctttccatag Has a 5′ G for U6 expression 6Cmismatch distance aatgttctaaacCatcctgc(SEQ ID NO: 388) Tiling 50 nt 6gcaggttcctggaactggaatctctttccat Has a 5′ G for U6 expression 6Cmismatch distance agaatgttctaaacCatcct(SEQ ID NO; 389) Tiling 50 nt 4gaccaggttcctggaactggaatctctacca Has a 5′ G for U6 expression 6Cmismatch distance tagaatgttctaaacCatc(SEQ ID NO: 390) Tiling 50 nt 2ggtaccaggttcctggaactggaatctcttt Has a 5′ G for U6 expression 6Cmismatch distance ccatagaatgttctaaacCa(SEQ ID NO: 391) Tiling 70 nt 70gCatcctgcggcctctactctgcattcaatt Has a 5′ G for U6 expression 6Cmismatch distance acatactgacacattcggcaacatgtttttcctggtttat(SEQ ID NO: 392) Tiling 70 nt 68gacCatcctgcggcctctactctgcattcaa Has a 5′ G for U6 expression 6Cmismatch distance ttacatactgacacattcggcaacatgtttttcctggttt(SEQ ID NO: 393) Tiling 70 nt 66gaaacCatcctgcggcctctactctgcattc Has a 5′ G for U6 expression 6Cmismatch distance aattacatactgacacattcggcaacatgtttttcctggt(SEQ ID NO: 394) Tiling 70 nt 64gctaaacCatcctgcggcctctactctgcat Has a 5′ G for U6 expression 6Cmismatch distance tcaattacatactgacacattcggcaacatgtttttcctg(SEQ ID NO: 395) Tiling 70 nt 62gttctaaacCatcctgcggcctctactctgc Has a 5′ G for U6 expression 6Cmismatch distance attcaattacatactgacacattcggcaacatgtttttcc(SEQ ID NO: 396) Tiling 70 nt 60gtgttctaaacCatcctgcggcctctactct Has a 5′ G for U6 expression 6Cmismatch distance gcattcaattacatactgacacattcggcaacatgttttt(SEQ ID NO: 397) Tiling 70 nt 58gaatgttctaaacCatcctgcggcctctact Has a 5′ G for U6 expression 6Cmismatch distance ctgcattcaattacatactgacacattcggcaacatgttt(SEQ ID NO: 398) Tiling 70 nt 56gagaatgttctaaacCatcctgcggcctcta Has a 5′ G for U6 expression 6Cmismatch distance ctctgcattcaattacatactgacacattcggcaacatgt(SEQ ID NO: 399) Tiling 70 nt 54gatagaatgttctaaacCatcctgcggcctc Has a 5′ G for U6 expression 6Cmismatch distance tactctgcattcaattacatactgacacattcggcaacat(SEQ ID NO: 400) Tiling 70 nt 52gccatagaatgttctaaacCatcctgcggcc Has a 5′ G for U6 expression 6Cmismatch distance tctactctgcattcaattacatactgacacattcggcaac(SEQ ID NO: 401) Tiling 70 nt 50gttccatagaatgttctaaacCatcctgcgg Has a 5′ G for U6 expression 6Cmismatch distance cctctactctgcattcaattacatactgacacattcggca(SEQ ID NO: 402) Tiling 70 nt 48gctaccatagaatgttctaaacCatcctgcg Has a 5′ G for U6 expression 6Cmismatch distance gcctctactctgcattcaattacatactgacacattcgg(SEQ ID NO: 403) Tiling 70 nt 46 gctctaccatagaatgttctaaacCatcctgHas a 5′ G for U6 expression 6C mismatch distancecggcctctactctgcattcaattacatactg acacattc(SEQ ID NO: 404) Tiling 70 nt 44gatctctaccatagaatgttctaaacCatcc Has a 5′ G for U6 expression 6Cmismatch distance tgcggcctctactctgcattcaattacatactgacacat(SEQ ID NO: 405) Tiling 70 nt 42 ggaatctctaccatagaatgttctaaacCatHas a 5′ G for U6 expression 6C mismatch distancecctgcggcctctactctgcattcaattacat actgacac(SEQ ID NO: 406) Tiling 70 nt 40gtggaatctctaccatagaatgttctaaacC Has a 5′ G for U6 expression 6Cmismatch distance atcctgcggcctctactctgcattcaattacatactgac(SEQ ID NO: 407) Tiling 70 nt 38 gactggaatctctaccatagaatgttctaaaHas a 5′ G for U6 expression 6C mismatch distancecCatcctgcggcctctactctgcattcaatt acatactg(SEQ ID NO: 408) Tiling 70 nt 36ggaactggaatctctaccatagaatgttcta Has a 5′ G for U6 expression 6Cmismatch distance aacCatcctgcggcctctactctgcattcaattacatac(SEQ ID NO: 409) Tiling 70 nt 34 gtggaactggaatctctttccatagaatgttHas a 5′ G for U6 expression 6C mismatch distancectaaacCatcctgcggcctctactctgcatt caattacat(SEQ ID NO: 410)Tiling 70 nt 32 gcctggaactggaatctctttccatagaatgHas a 5′ G for U6 expression 6C mismatch distancettctaaacCatcctgcggcctctactctgca ttcaattac(SEQ ID NO: 411)Tiling 70 nt 30 gttcctggaactggaatctattccatagaatHas a 5′ G for U6 expression 6C mismatch distancegttctaaacCatcctgcggcctctactctgc attcaaft(SEQ ID NO: 412) Tiling 70 nt 28gggttcctggaactggaatctctttccatag Has a 5′ G for U6 expression 6Cmismatch distance aatgttctaaacCatcctgcggcctctactctgcattcaa(SEQ ID NO: 413) Tiling 70 nt 26gcaggttcctggaactggaatctctttccat Has a 5′ G for U6 expression 6Cmismatch distance agaatgttctaaacCatcctgcggcctctactctgcattc(SEQ ID NO: 414) Tiling 70 nt 24gaccaggttcctggaactggaatctctttcc Has a 5′ G for U6 expression 6Cmismatch distance atagaatgttctaaacCatcctgcggcctctactctgcat(SEQ ID NO: 415) Tiling 70 nt 22ggtaccaggttcctggaactggaatctcttt Has a 5′ G for U6 expression 6Cmismatch distance ccatagaatgttctaaacCatcctgcggcctctactctgc(SEQ ID NO: 416) Tiling 70 nt 20gatgtaccaggttcctggaactggaatctat Has a 5′ G for U6 expression 6Cmismatch distance tccatagaatgttctaaacCatcctgcggcctctactct(SEQ ID NO: 417) Tiling 70 nt 18 ggtatgtaccaggttcctggaactggaatctHas a 5′ G for U6 expression 6C mismatch distancectttccatagaatgttctaaacCatcctgcg gcctctact(SEQ ID NO: 418)Tiling 70 nt 16 gacgtatgtaccaggttcctggaactggaatHas a 5′ G for U6 expression 6C mismatch distancectctttccatagaatgttctaaacCatcctg cggcctcta(SEQ ID NO: 419)Tiling 70 nt 14 gacacgtatgtaccaggttcctggaactggaHas a 5′ G for U6 expression 6C mismatch distanceatctattccatagaatgttctaaacCatcct gcggcctc(SEQ ID NO: 420) Tiling 70 nt 12gcaacacgtatgtaccaggttcctggaactg Has a 5′ G for U6 expression 6Cmismatch distance gaatctctttccatagaatgttctaaacCatcctgcggcc(SEQ ID NO: 421) Tiling 70 nt 10gcccaacacgtatgtaccaggttcctggaac Has a 5′ G for U6 expression 6Cmismatch distance tggaatctctttccatagaatgttctaaacCatcctgcgg(SEQ ID NO: 422) Tiling 70 nt 8 ggacccaacacgtatgtaccaggttcctggaHas a 5′ G for U6 expression 6C mismatch distanceactggaatctctttccatagaatgttctaaa cCatcctgc(SEQ ID NO: 423) Tiling 70 nt 6gttgacccaacacgtatgtaccaggttcctg Has a 5′ G for U6 expression 6Cmismatch distance gaactggaatctctttccatagaatgttctaaacCatcct(SEQ ID NO: 424) Tiling 70 nt 4 gccttgacccaacacgtatgtaccaggttccHas a 5′ G for U6 expression 6C mismatch distancetggaactggaatctctttccatagaatgttc taaacCatc(SEQ ID NO: 425) Tiling 70 nt 2gttccttgacccaacacgtatgtaccaggtt Has a 5′ G for U6 expression 6Cmismatch distance cctggaactggaatctctttccatagaatgttctaaacCa(SEQ ID NO: 426) Tiling 84 nt 84gCatcctgcggcctctactctgcattcaatt Has a 5′ G for U6 expression 6Cmismatch distance acatactgacacattcggcaacatgtttttcctggtttattttcacacagtcca(SEQ ID NO: 427) Tiling 84 nt 82gacCatcctgcggcctctactctgcattcaa Has a 5′ G for U6 expression 6Cmismatch distance ttacatactgacacattcggcaacatgtttttcctggtttattttcacacagtc(SEQ ID NO: 428) Tiling 84 nt 80gaaacCatcctgcggcctctactctgcattc Has a 5′ G for U6 expression 6Cmismatch distance aattacatactgacacattcggcaacatgcctggtttattttcacacag(SEQ ID NO: 429) Tiling 84 nt 78gctaaacCatcctgcggcctctactctgcat Has a 5′ G for U6 expression 6Cmismatch distance tcaattacatactgacacattcggcaacatgtttttcctggtttattttcacac(SEQ ID NO: 430) Tiling 84 nt 76gttctaaacCatcctgcggcctctactctgc Has a 5′ G for U6 expression 6Cmismatch distance attcaattacatactgacacattcggcaacatgtttttcctggtttattttcac(SEQ ID NO: 431) Tiling 84 nt 74gtgttctaaacCatcctgcggcctctactct Has a 5′ G for U6 expression 6Cmismatch distance gcattcaattacatactgacacattcggcaacatgtttttcctggtttattttc(SEQ ID NO: 432) Tiling 84 nt 72gaatgttctaaacCatcctgcggcctctact Has a 5′ G for U6 expression 6Cmismatch distance ctgcattcaattacatactgacacattcggcaacatgtttttcctggtttattt(SEQ ID NO: 433) Tiling 84 nt 70gagaatgttctaaacCatcctgcggcctcta Has a 5′ G for U6 expression 6Cmismatch distance ctctgcattcaattacatactgacacattcggcaacatgtttttcctggtttat(SEQ ID NO: 434) Tiling 84 nt 68gatagaatgttctaaacCatcctgcggcctc Has a 5′ G for U6 expression 6Cmismatch distance tactctgcattcaattacatactgacacattcggcaacatgtttttcctggttt(SEQ ID NO: 435) Tiling 84 nt 66gccatagaatgttctaaacCatcctgcggcc Has a 5′ G for U6 expression 6Cmismatch distance tctactctgcattcaattacatactgacacattcggcaacatgtttttcctggt(SEQ ID NO: 436) Tiling 84 nt 64gttccatagaatgttctaaacCatcctgcgg Has a 5′ G for U6 expression 6Cmismatch distance cctctactctgcattcaattacatactgacacattcggcaacatgtttttcctg(SEQ ID NO: 437) Tiling 84 nt 62gctttccatagaatgttctaaacCatcctgc Has a 5′ G for U6 expression 6Cmismatch distance ggcctctactctgcattcaattacatactgacacattcggcaacatgtttttcc(SEQ ID NO: 438) Tiling 84 nt 60gctctttccatagaatgttctaaacCatcct Has a 5′ G for U6 expression 6Cmismatch distance gcggcctctactctgcattcaattacatactgacacattcggcaacatgttttt(SEQ ID NO: 439) Tiling 84 nt 58gatctctttccatagaatgttctaaacCatc Has a 5′ G for U6 expression 6Cmismatch distance ctgcggcctctactctgcattcaattacatactgacacattcggcaacatgttt(SEQ ID NO: 440) Tiling 84 nt 56ggaatctattccatagaatgttctaaacCat Has a 5′ G for U6 expression 6Cmismatch distance cctgcggcctctactctgcattcaattacatactgacacattcggcaacatgt(SEQ ID NO: 441) Tiling 84 nt 54gtggaatctctttccatagaatgttctaaac Has a 5′ G for U6 expression 6Cmismatch distance Catcctgcggcctctactctgcattcaattacatactgacacattcggcaacat(SEQ ID NO: 442) Tiling 84 nt 52gactggaatctctaccatagaatgttctaaa Has a 5′ G for U6 expression 6Cmismatch distance cCatcctgcggcctctactctgcattcaattacatactgacacattcggcaac(SEQ ID NO: 443) Tiling 84 nt 50ggaactggaatctctaccatagaatgttcta Has a 5′ G for U6 expression 6Cmismatch distance aacCatcctgcggcctctactctgcattcaattacatactgacacattcggca(SEQ ID NO: 444) Tiling 84 nt 48Has a 5′ G for U6 expression 6C mismatch distancegtggaactggaatctctttccatagaatgtt ctaaacCatcctgcggcctctactctgcattcaattacatactgacacattcgg(SEQ ID NO: 445) Tiling 84 nt 46gcctggaactggaatctctttccatagaatg Has a 5′ G for U6 expression 6Cmismatch distance ttctaaacCatcctgcggcctctactctgcattcaattacatactgacacattc(SEQ ID NO: 446) Tiling 84 nt 44gttcctggaactggaatctctaccatagaat Has a 5′ G for U6 expression 6Cmismatch distance gttctaaacCatcctgcggcctctactctgcattcaattacatactgacacat(SEQ ID NO: 447) Tiling 84 nt 42gggttcctggaactggaatctctttccatag Has a 5′ G for U6 expression 6Cmismatch distance aatgttctaaacCatcctgcggcctctactctgcattcaattacatactgacac(SEQ ID NO: 448) Tiling 84 nt 40gcaggttcctggaactggaatctctttccat Has a 5′ G for U6 expression 6Cmismatch distance agaatgttctaaacCatcctgcggcctctactctgcattcaattacatactgac(SEQ ID NO: 449) Tiling 84 nt 38gaccaggttcctggaactggaatctctacca Has a 5′ G for U6 expression 6Cmismatch distance tagaatgttctaaacCatcctgcggcctctactctgcattcaattacatactg(SEQ ID NO: 450) Tiling 84 nt 36ggtaccaggttcctggaactggaatctcttt Has a 5′ G for U6 expression 6Cmismatch distance ccatagaatgttctaaacCatcctgcggcctctactctgcattcaattacatac(SEQ ID NO: 451) Tiling 84 nt 34gatgtaccaggttcctggaactggaatctct Has a 5′ G for U6 expression 6Cmismatch distance accatagaatgttctaaacCatcctgcggcctctactctgcattcaattacat(SEQ ID NO: 452) Tiling 84 nt 32ggtatgtaccaggttcctggaactggaatct Has a 5′ G for U6 expression 6Cmismatch distance ctttccatagaatgttctaaacCatcctgcggcctctactctgcattcaattac(SEQ ID NO: 453) Tiling 84 nt 30gacgtatgtaccaggttcctggaactggaat Has a 5′ G for U6 expression 6Cmismatch distance ctctaccatagaatgttctaaacCatcctgcggcctctactctgcattcaatt(SEQ ID NO: 454) Tiling 84 nt 28gacacgtatgtaccaggttcctggaactgga Has a 5′ G for U6 expression 6Cmismatch distance atctctaccatagaatgttctaaacCatcctgcggcctctactctgcattcaa(SEQ ID NO: 455) Tiling 84 nt 26gcaacacgtatgtaccaggttcctggaactg Has a 5′ G for U6 expression 6Cmismatch distance gaatctctttccatagaatgttctaaacCatcctgcggcctctactctgcattc(SEQ ID NO: 456) Tiling 84 nt 24gcccaacacgtatgtaccaggttcctggaac Has a 5′ G for U6 expression 6Cmismatch distance tggaatctattccatagaatgttctaaacCatcctgcggcctctactctgcat(SEQ ID NO: 457) Tiling 84 nt 22ggacccaacacgtatgtaccaggttcctgga Has a 5′ G for U6 expression 6Cmismatch distance actggaatctctttccatagaatgttctaaacCatcctgcggcctctactctgc(SEQ ID NO: 458) Tiling 84 nt 20gttgacccaacacgtatgtaccaggttcctg Has a 5′ G for U6 expression 6Cmismatch distance gaactggaatctctttccatagaatgttctaaacCatcctgcggcctctactct(SEQ ID NO: 459) Tiling 84 nt 18gccttgacccaacacgtatgtaccaggttcc Has a 5′ G for U6 expression 6Cmismatch distance tggaactggaatctctttccatagaatgttctaaacCatcctgcggcctctact(SEQ ID NO: 460) Tiling 84 nt 16gttccttgacccaacacgtatgtaccaggtt Has a 5′ G for U6 expression 6Cmismatch distance cctggaactggaatctctttccatagaatgttctaaacCatcctgcggcctcta(SEQ ID NO: 545) Tiling 84 nt 14gggttccttgacccaacacgtatgtaccagg Has a 5′ G for U6 expression 6Cmismatch distance ttcctggaactggaatctctttccatagaatgttctaaacCatcctgcggcctc(SEQ ID NO: 461) Tiling 84 nt 12gttggttccttgacccaacacgtatgtacca Has a 5′ G for U6 expression 6Cmismatch distance ggttcctggaactggaatctctttccatagaatgttctaaacCatcctgcggcc(SEQ ID NO: 462) Tiling 84 nt 10gccttggttccttgacccaacacgtatgtac Has a 5′ G for U6 expression 6Cmismatch distance caggttcctggaactggaatctattccatagaatgttctaaacCatcctgcgg(SEQ ID NO: 463) Tiling 84 nt 8ggcccttggttccttgacccaacacgtatgt Has a 5′ G for U6 expression 6Cmismatch distance accaggttcctggaactggaatctattccatagaatgttctaaacCatcctgc(SEQ ID NO: 464) Tiling 84 nt 6gccgccatggttccttgacccaacacgtatg Has a 5′ G for U6 expression 6Cmismatch distance taccaggttcctggaactggaatctctttccatagaatgttctaaacCatcct(SEQ ID NO: 465) Tiling 84 nt 4gcgccgcccttggttccttgacccaacacgt Has a 5′ G for U6 expression 6Cmismatch distance atgtaccaggttcctggaactggaatctctttccatagaatgttctaaacCatc(SEQ ID NO: 466) Tiling 84 nt 2ggtcgccgcccttggttccttgacccaacac Has a 5′ G for U6 expression 6Cmismatch distance gtatgtaccaggttcctggaactggaatctcatttcctagaatgttctaaacCa(SEQ ID NO: 467) ADAR non-GTAATGCCTGGCTTGTCGACGCATAGTCTG Has a 5′ G for U6 expression 6Ctargeting guide (SEQ ID NO: 468) PFS binding screengaaaacgcaggttcctcCagtttcgggagca Has a 5′ G for U6 expression 7Bguide for TAG gcgcacgtctccctgtagtc(SEQ ID motif NO: 469)PFS binding screen  gacgcaggttcctctagCttcgggagcagcgHas a 5′ G for U6 expression 7B guide for AACcacgtctccctgtagtcaag(SEQ ID motif NO: 470) PFS binding screenGTAATGCCTGGCTTGTCGACGCATAGTCTG Has a 5′ G for U6 expression 7Bnon-targeting (SEQ ID NO: 471) Motif preferencegatagaatgactaaacCatcctgcggcctct Has a 5′ G for U6 expression 7Ctargeting guide actctgcattcaattacat(SEQ ID NO: 472) Motif preferenceGTAATGCCTGGCTTGTCGACGCATAGTCTG Has a 5′ G for U6 expression 7Cnon-targeting (SEQ ID NO: 473) guide PPIB tiling guidegCaaggccacaaaattatccactgggaacag Has a 5′ G for U6 expression 13D 50 mismatch tctaccgaagagac(SEQ ID NO: distance 474) PPIB tiling guidegcctgtagcCaaggccacaaaattatccact Has a 5′ G for U6 expression 13D 42 mismatch gtttttggaacagtctacc(SEQ ID dtstance NO: 475)PPIB tiling guide gctactctcctgtagcCaaggccacaaaattHas a 5′ G for U6 expression 13D  34 mismatch atccactgtttttggaaca(SEQ IDdistance NO: 476) PPIB tiling guide ggccaaatcctactctcctgtagcCaaggccHas a 5′ G for U6 expression 13D  26 mismatchacaaaattatccactgta(SEQ ID NO: distance 477) PPIB tiling guidegtttttgtagccaaatcctttctctcctgta Has a 5′ G for U6 expression 13D 18 mismatch gcCaaggccacaaaattatc(SEQ ID NO: distance 478)PPIB tiling guide gatagctgtattgtagccaaatcctactctcHas a 5′ G for U6 expression 13D  10 mismatchctgtagcCaaggccaca(SEQ ID NO: distance 479) PPIB tiling guide 2gacgatggaatttgctggtagccaaatcctt Has a 5′ G for U6 expression 13D mismatch distance tctctcctgtagcCa(SEQ ID NO: 480) Targeting guide,gatagaatgactaaacGatcctgcggcctct Has a 5′ G for U6 expression 13D opposite base G actctgcattcaattacat(SEQ ID NO: 481) Targeting guide,gatagaatgactaaacAatcctgcggcctct Has a 5′ G for U6 expression 13D opposite base A actctgcattcaattacat(SEQ ID NO: 482) Targeting guide,gatagaatgactaaacTatcctgcggcctct Has a 5′ G for U6 expression 13D opposite base C actctgcattcaattacat(SEQ ID NO: 483) AVPR2 guide 37ggtcccacgcggccCacagctgcaccaggaa Has a 5′ G for U6 expression 8Amismatch distance gaagggtgcccagcacagca(SEQ ID NO: 484) AVPR2 guide 35ggggtcccacgcggccCacagctgcaccagg Has a 5′ G for U6 expression 8Amismatch distance aagaagggtgcccagcacag(SEQ ID NO: 485) AVPR2 guide 33gccgggtcccacgcggccCacagctgcacca Has a 5′ G for U6 expression 8Amismatch distance ggaagaagggtgcccagcac(SEQ ID NO: 486) guide 37gggtgatgacatccCaggcgatcgtgtggcc Has a 5′ G for U6 expression 8Bmismatch distance tccaggagcccagagcagga(SEQ ID NO: 487) FANCC guide 35gagggtgatgacatccCaggcgatcgtgtgg Has a 5′ G for U6 expression 8Bmismatch distance cctccaggagcccagagcag(SEQ ID NO: 488) FANCC guide 32gatcagggtgatgacatccCaggcgatcgtg Has a 5′ G for U6 expression 8Bmismatch distance tggcctccaggagcccagag(SEQ ID NO: 489) Synthetic diseaseggtggctccattcactcCaatgctgagcact Has a 5′ G for U6 expression 8Egene target IL2RG tccacagagtgggttaaagc(SEQ ID NO: 490) Synthetic diseasegtttctaatatagCcagactgatggactatt Has a 5′ G for U6 expression 8Egene target F8 ctcaattaataatgat(SEQ ID NO: 491) Synthetic diseasegagatgttgctgtggatCcagtccacagcca Has a 5′ G for U6 expression 8Egene target LDLR gcccgtcgggggcctggatg(SEQ ID NO: 492) Synthetic diseasegcaggccggcccagctgCcaggtgcacctgc Has a 5′ G for U6 expression 8Egene target CBS tcggagcatcgggccggatc(SEQ ID NO: 493) Synthetic diseasegcaaagaacctctgggtCcaagggtagacca Has a 5′ G for U6 expression 8Egene target HBB ccagcagcctgcccagggcc(SEQ ID NO: 494) Synthetic diseasegaagagaaacttagtttCcagggctttggta Has a 5′ G for U6 expression 8Egene target gagggcaaaggttgatagca(SEQ ID NO: ALDOB 495) Synthetic diseasegtcagcctagtgcagagCcactggtagttgg Has a 5′ G for U6 expression 8Egene target DMD tggttagagtttcaagttcc(SEQ ID NO: 496) Synthetic diseaseggctcattgtgaacaggCcagtaatgtccgg Has a 5′ G for U6 expression 8Egene target gatggggcggcataggcggg(SEQ ID NO: SMAD4 497) Synthetic diseasegtagctaaagaacttgaCcaagacatatcag Has a 5′ G for U6 expression 8Egene target BRCA2 gatccacctcagctcctaga(SEQ ID NO: 498) Synthetic diseaseggggcattgttctgtgcCcagtcctgctggt Has a 5′ G for U6 expression 8Egene target agacctgctccccggtggct(SEQ ID NO: GRIN2A 499)Synthetic disease gagaagtcgttcatgtgCcaccgtgggagcgHas a 5′ G for U6 expression 8E gene target SCN9Atacagtcatcattgatcttg(SEQ ID NO: 500) Synthetic diseasegggattaatgctgaacgCaccaaagttcatc Has a 5′ G for U6 expression 8Egene target ccaccacccatattactacc(SEQ ID NO: TARDBP 546)Synthetic disease gctccaaaggctttcctCcactgttgcaaagHas a 5′ G for U6 expression 8E gene target CFTRttattgaatcccaagacaca(SEQ ID NO: 501) Synthetic diseasegatgaatgaacgatttcCcagaactccctaa Has a 5′ G for U6 expression 8Egene target UBE3A tcagaacagagtccctggta(SEQ ID NO: 502) Synthetic diseaseggagcctctgccggagcCcagagaacccgag Has a 5′ G for U6 expression 8Egene target SMPD1 agtcagacagagccagcgcc(SEQ ID NO: 503) Synthetic diseaseggottccgtggagacacCcaatcaatttgaa Has a 5′ G for U6 expression 8Egene target USH2A gagatcttgaagtgatgcca(SEQ ID NO: 504) Synthetic diseasegtgggactgccctcctcCcatttgcagatgc Has a 5′ G for U6 expression 8Egene target MEN1 cgtcgtagaatcgcagcagg(SEQ ID NO: 505) Synthetic diseasegcttcttcaatagttctCcagctacactggc Has a 5′ G for U6 expression 8Egene target aggcatatgcccgtgttcct(SEQ ID NO: C8orf37 506)Synthetic disease gattccttttcttcgtcCcaattcacctcagHas a 5′ G for U6 expression 8E gene target MLH1tggctagtcgaagaatgaag(SEQ ID NO: 507) Synthetic diseasegcagcttcagcaccttcCagtcagactcctg Has a 5′ G for U6 expression 8Egene target TSC2 cttcaagcactgcagcagga(SEQ ID NO: 508) Synthetic diseasegccatttgcttgcagtgCcactccagaggat Has a 5′ G for U6 expression 8Egene target NF1 tccggattgccataaatact(SEQ ID NO: 509) Synthetic diseasegttcaatagttaggtcCagtatcgtnacagc Has a 5′ G for U6 expression 8Egene target MSH6 ccttcttggtagatttca(SEQ ID NO: 510) Synthetic diseaseggcaaccgtcttctgacCaaatggcagaaca Has a 5′ G for U6 expression 8Egene target SMN1 tttgtccccaactttccact(SEQ ID NO: 511) Synthetic diseasegcgactttccaatgaacCactgaagcccagg Has a 5′ G for U6 expression 8Egene target tatgacaaagccgatgatct(SEQ ID NO: SH3TC2 512)Synthetic disease gtttacactcatgcttcCacagctttaacagHas a 5′ G for U6 expression 8E gene targetatcatttggttccttgatga(SEQ ID NO: DNAH5 513) Synthetic diseasegcttaagcttccgtgtcCagccttcaggcag Has a 5′ G for U6 expression 8Egene target MECP2 ggtggggtcatcatacatgg(SEQ ID NO: 514) Synthetic diseaseggacagctgggctgatcCatgatgtcatcca Has a 5′ G for U6 expression 8Egene target gaaacactggggaccctcag(SEQ ID NO: ADGRV1 515)Synthetic disease gtctcatctcaactttcCatatccgtatcatHas a 5′ G for U6 expression 8E gene target AHI1ggaatcatagcatcctgtaa(SEQ ID NO: 516) Synthetic diseasegcatgcagacgcggttcCactcgcagccaca Has a 5′ G for U6 expression 8Egene target PRKN gttccagcaccactcgagcc(SEQ ID NO: 517) Synthetic diseasegttggttagggtcaaccCagtattctccact Has a 5′ G for U6 expression 8Egene target cttgagttcaggatggcaga(SEQ ID NO: COL3A1 518)Synthetic disease gctacactgtccaacacCcactctcgggtcaHas a 5′ G for U6 expression 8E gene target BRCA1ccacaggtgcctcacacatc(SEQ ID NO: 519) Synthetic diseasegctgcactgtgtaccccCagagctccgtgtt Has a 5′ G for U6 expression 8Egene target gccgacatcctggggtggct(SEQ ID NO: MYBPC3 520)Synthetic disease gagcttcctgccactccCaacaggtttcacaHas a 5′ G for U6 expression 8E gene target APCgtaagcgcgtatctgttcca(SEQ ID NO: 521) Synthetic diseasegacggcaagagcttaccCagtcacttgtgtg Has a 5′ G for U6 expression 8Egene target BMPR2 gagacttaaatacttgcata(SEQ ID NO: 522) KRAS tiling guidegCaaggccacaaaattatccactgggaacag Has a 5′ G for U6 expression 9A50 mismatch tctaccgaagagac(SEQ ID NO: 523) distance KRAS tiling guidegcctgtagcCaaggccacaaaattatccact Has a 5′ G for U6 expression 9A42 mismatch gtttttggaacagtctacc(SEQ ID NO: distance 524)KRAS tiling guide gctactctcctgtagcCaaggccacaaaattHas a 5′ G for U6 expression 9A 34 mismatchatccactgtttttggaaca(SEQ ID NO: distance 525) KRAS tiling guideggccaaatcctttctctcctgtagcCaaggc Has a 5′ G for U6 expression 9A26 mismatch cacaaaattatccactgttt(SEQ ID NO: distance 526)KRAS tiling guide gtttttgtagccaaatcctttctctcctgtaHas a 5′ G for U6 expression 9A 18 mismatchgcCaaggccacaaaattatc(SEQ ID NO: distance 527) KRAS tiling guidegatttgctgttatgtagccaaatcctactct Has a 5′ G for U6 expression 9A10 mismatch cctgtagcCaaggccaca(SEQ ID NO: distance 528)KRAS tiling guide gacgatggaatttgctggtagccaaatccttHas a 5′ G for U6 expression 9A 2 mismatchtctctcctgtagcCa(SEQ ID NO: 529) distance KRAS tiling non-GTAATGCCTGGCTTGTCGACGCATAGTCTG Has a 5′ G for U6 expression 9Atargeting guide (SEQ ID NO: 530) Luciferase W85XgatagaatgttctaaacCatcctgcggcctc Has a 5′ G for U6 expression 53B targeting guide tactctgcattcaattacat(SEQ ID NO: for 531) transcriptomespecificity Non-targeting GCAGGGTTTTCCCAGTCACGACGTTGTAAAGHas a 5′ G for U6 expression 9C guide for TTG(SEQ ID NO: 532)transcriptome specificity endogenous KRASgtcaaggcactcttgccCacgccaccagctc Has a 5′ G for U6 expression 10F guide 2 caactaccacaagtttatat(SEQ ID NO: 533) endogenous PPIBgcaaagatcacccggccCacatcttcatctc Has a 5′ G for U6 expression 10G guide 1 caattcgtaggtcaaaatac(SEQ ID NO: 534) endogenous KRASGcgccaccagctccaacCaccacaagtttat Has a 5′ G for U6 expression 10F guide 1 attcagtcattttcagcagg(SEQ ID NO: 535) endogenous KRASGtactccatcaattacCacttgcttcctgta Has a 5′ G for U6 expression 10F guide 3 ggaatcctctattGTtgga(SEQ ID NO: 536) endogenous PPIBGctactctcctgtagcCaaggccacaaaatt Has a 5′ G for U6 expression 10G guide 2 atccactgtttttggaaca(SEQ ID NO: 537) endogenous non-GTAATGCCTGGCTTGTCGACGCATAGTCTG Has a 5′ G for U6 expression 10F targeting guide (SEQ ID NO: 538) BoxB Cluc guidetctaccataGGCCCTGAAAAAGGGCCtgttc Has a 5′ G for U6 expression 18B taaacCatcctgcggcctctactcGGCCCTG AAAAAGGGCCattcaattac(SEQ ID NO: 539)BoxB cagctggcgaGGCCCTGAAAAAGGGCCgggg Has a 5′ G for U6 expression 18B non-targeting atgtgcCgcaaggcgattaagttggGGCCCT guideGAAAAAGGGCCacgccagggt(SEQ ID NO: 540) StafforstGTGGAATAGTATAACAATATGCTAAATGTTG Has a 5′ G for U6 expression 18C full length TTATAGTATCCCACtctaaaCCAtcctgcgG ADAR2 guide 1GGCCCTCTTCAGGGCCC(SEQ ID NO: 541) Stafforst fullGTGGAATAGTATAACAATATGCTAAATGTTG Has a 5′ G for U6 expression 18C length ADAR2 TTATAGTATCCCACaccctggcgttacccaG non-targetingGGCCCTCTTCAGGGCCC(SEQ ID NO: guide 542)

REFERENCES

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Example 4—Additional Type VI-B Effectors

TABLE 13 Paludibactermktsanniyfnginsfkkifdskgaiapiaekscrnfdikaqndvnkegrihyfavghtfpropionicigeneskqldtenlfeyvldenlrakrptrfislqqfdkefienikrlisdirninshyihrfdpl WB4kidavptniidflkesfelaviqiylkekginylqfsenphadqklvaflhdkflpldek(NC_014734.1)ktsmlqnetpqlkeykeyrkyfktlskqaaidqllfaeketdyiwnlfdshpvltisagk >WP_013446107ylsfysclfllsmflykseangliskikgfkkntteeekskreiftffskrfnsmdidseenqlvkfrdlilylnhypvawnkdleldssnpamtdklkskiieleinrsfplyegnerfatfakyqiwgkkhlgksiekeyinasftdeeitaytyetdtcpelkdahkkladlkaakglfgkrkeknesdikktetsirelqhepnpikdkliqrieknlltvsygrnqdrfmdfsarflaeinyfgqdasfkmyhfyatdeqnselekyelpkdkkkydslkfhqgklvhfisykehlkryeswddafviennaiqlklsfdgventvtiqralliylledalrniqnntaenagkqllqeyyshnkadlsafkqiltqqdsiepqqktefkkllprrllnnyspainhlqtphsslplilekallaekrycslvvkakaegnyddfikrnkgkqfklqfirkawnlmyfrnsylqnvqaaghhksfhierdefndfsrymfafeelsqykyylnemfekkgffennefkilfqsgtslenlyektkqkfeiwlasntaktnkpdnyhlnnyeqqfsnqlffinlshfinylkstgklqtdangqiiyealnnvqylipeyyytdkpersesksgnklynklkatkledallyemamcylkadkqiadkakhpitklltsdvefnitnkegiqlyhllvpfkkidafiglkmhkeqqdkkhptsflanivnylelvkndkdirktyeafstnpvkrtltyddlakidghlisksikftnvtleleryfifkeslivkkgnnidfkyikglrnyynnekkknegirnkafhfgipdsksydglirdaevmfianevkpthatkytdlnkqlhtvcdklmetvhndyfskegdgkkkreaagqkyfeniisak (SEQ ID NO: 547) Prevotellamnipalvenqkkyfgtysvmamlnaqtvldhiqkvadiegegnennenlwfhpvmshlyn sp. P5-60akngydkqpektmfiierlqsyfpflkimaenqreysngkykqnrvevnsndifevlkra(NZ_JXQJ01000080.1)fgvlkmyrdltnhyktyeeklidgcefltsteqpfsgmiskyytvalrntkerygykaed >WP_044074780.1lafiqdnrykftkdaygkrksqvntgsflslqdyngdttkklhlsgvgialliclfldkqyinlflsrlpifssynaqseerriiirsfginsikqpkdrihseksnksvamdmlnevkrcpdelfttlsaekqsrfriisddhnevlmkrssdrfvplllqyidygklfdhirfhvnmgklryllkadktcidgqtrvrvieqpingfgrleevetmrkqengtfgnsgirirdfenmkrddanpanypyivetythyilennkvemfisdeenptpllpvieddryvvktipscrmstleipamafhmflfgsektekliidvhdrykrlfqamqkeevtaeniasfgiaesdlpqkimdlisgnahgkdvdafirltvddmltdterrikrfkddrksirsadnkmgkrgfkgistgkladflakdivlfqpsvndgenkitglnyrimqsaiavydsgddyeakqqfklmfekarligkgttephpflykvfvrsipanavdfyerylierkfyliglsneikkgnrvdvpfirrdqnkwktpamktlgriysedlpvelprqmfdneikshlkslpqmegidfnnanvtyliaeymkrvinddfqtfyqwkrnyrymdmlrgeydrkgslqhcftsieereglwkerasrteryrklasnkirsnrqmrnasseeietildkrlsncrneyqksekiirryrvqdallfllakktlteladfdgerfklkeimpdaekgilseimpmsftfekggkiytitsggmklknygdffvlasdkrignllelvgsntvskedimeefkkydqcrpeissivfnlekwafdtypelparydrkekvdfwsildvlsnnkdinneqsyilrkirnafdhnnypdkgiveikalpeiamsikkafgeyaimk (SEQ ID NO: 548) Prevotellamnipalvenqkkyfgtysvmamlnaqtvldhiqkvadiegegnennenlwfhpvmshlyn sp. P4-76akngydkqpektmfiierlqsyfpflkimaenqreysngkykqnrvevnsndifevlkra(NZ_JXQ101000021.1)fgvlkmyrdqashyktydeklidgcefltsteqplsgminnyytvalrnmnerygykted >WP_044072147.1lafiqdkrfkfvkdaygkkksqvntgfflslqdyngdtqkklhlsgvgialliclfldkqyiniflsrlpifssynaqseerriiirsfginsikqpkdrihseksnksvamdmlneikrcpnelfetlsaekqsrfriisndhnevlmkrssdrfvplllqyidygklfdhirfhvnmgklryllkadktcidgqtrvrvieqpingfgrleevetmrkqengtfgnsgirirdfenmkrddanpanypyivdtythyilennkvemfisdeetpapllpvieddryvvktipscrmstleipamafhmflfgskkteklivdvhnrykrlfkamqkeevtaeniasfgiaesdlpqkiidlisgnahgkdvdafirltvddmladterrikrfkddrksirsadnkmgkrgfkgistgkladflakdivlfqpsvndgenkitglnyrimqsaiavynsgddyeakqqfklmfekarligkgttephpflykvfvrsipanavdfyerylierkfyliglsneikkgnrvdvpfirrdqnkwktpamktlgriydedlpvelprqmfdneikshlkslpqmegidfnnanvtyliaeymkrvinddfqtfyqwkrnyrymdmlrgeydrkgslqscftsveereglwkerasrteryrklasnkirsnrqmrnasseeietildkrlsnsrneyqksekvirryrvqdallfllakktlteladfdgerfklkeimpdaekgilseimpmsftfekggkkytitsegmklknygdffvlasdkrignllelvgsdtvskedimeefkkydqcrpeissivfnlekwafdtypelsarydreekvdfksilkillnnkninkeqsdilrkirnafdhnnypdkgvveiralpeiamsikkafgeyaimk (SEQ ID NO: 549) Prevotellamnipalvenqkkyfgtysvmamlnaqtvldhiqkvadiegegnennenlwfhpvmshlyn sp. P5-125akngydkqpektmfiierlqsyfpflkimaenqreysngkykqnrvevnsndifevlkra(NZ_JXQL01000055.1)fgvlkmyrdltnhyktyeeklndgcefltsteqplsgminnyytvalrnmnerygykted >WP_044065294.1lafiqdkrfkfvkdaygkkksqvntgfflslqdyngdtqkklhlsgvgialliclfldkqyiniflsrlpifssynaqseerriiirsfginsiklpkdrihseksnksvamdmlnevkrcpdelfttlsaekqsrfriisddhnevlmkrssdrfvplllqyidygklfdhirfhvnmgklryllkadktcidgqtrvrvieqpingfgrleeaetmrkqengtfgnsgirirdfenmkrddanpanypyivdtythyilennkvemfindkedsapllpvieddryvvktipscrmstleipamafhmflfgskkteklivdvhnrykrlfqamqkeevtaeniasfgiaesdlpqkildlisgnahgkdvdafirltvddmltdterrikrfkddrksirsadnkmgkrgfkgistgkladflakdivlfqpsvndgenkitglnyrimqsaiavydsgddyeakqqfklmfekarligkgttephpflykvfarsipanavefyerylierkfyltglsneikkgnrvdvpfirrdqnkwktpamktlgriysedlpvelprqmfdneikshlkslpqmegidfnnanvtyliaeymkrvldddfqtfyqwnrnyrymdmlkgeydrkgslqhcftsveereglwkerasrteryrkgasnkirsnrqmrnasseeietildkrlsnsrneyqksekvirryrvqdallfllakktlteladfdgerfklkeimpdaekgilseimpmsftfekggkkytitsegmklknygdffvlasdkrignllelvgsdivskedimeefnkydqcrpeissivfnlekwafdtypelsarydreekvdfksilkillnnkninkeqsdilrkirnafdhnnypdkgvveikalpeiamsikkafgeyaimk (SEQ ID NO: 550) Prevotellamnipalvenqkkyfgtysvmamlnaqtvldhiqkvadiegegnennenlwfhpvmshlyn sp. P5-119akngydkqpektmfiierlqsyfpflkimaenqreysngkykqnrvevnsndifevlkra(NZ_JXQK01000043.1)fgvlkmyrdltnhyktyeeklidgcefltsteqplsgmiskyytvalrntkerygykted >WP_042518169.1lafiqdnikkitkdaygkrksqvntgfflslqdyngdtqkklhlsgvgialliclfldkqyiniflsrlpifssynaqseerriiirsfginsiklpkdrihseksnksvamdmlnevkrcpdelfttlsaekqsrfriisddhnevlmkrstdrfvplllqyidygklfdhirfhvnmgklryllkadktcidgqtrvrvieqpingfgrleeaetmrkqengtfgnsgirirdfenvkrddanpanypyivdtythyilennkvemfisdkgssapllplieddryvvktipscrmstleipamafhmflfgskkteklivdvhnrykrlfqamqkeevtaeniasfgiaesdlpqkildlisgnahgkdvdafirltvddmltdterrikrfkddrksirsadnkmgkrgfkgistgkladflakdivlfqpsvndgenkitglnyrimqsaiavydsgddyeakqqfklmfekarligkgttephpflykvfarsipanavdfyerylierkfyltglcneikrgnrvdvpfirrdqnkwktpamktlgriysedlpvelprqmfdneikshlkslpqmegidfnnanvtyliaeymkrvinddfqtfyqwkrnyhymdmlkgeydrkgslqhcftsveereglwkerasrteryrklasnkirsnrqmrnasseeietildkrlsncrneyqksekvirryrvqdallfllakktlteladfdgerfklkeimpdaekgilseimpmsftfekggkkytitsegmklknygdffvlasdkrignllelvgsdivskedimeefnkydqcrpeissivfnlekwafdtypelsarydreekvdfksilkillnnkninkeqsdilrkirnafdhnnypdkgiveikalpeiamsikkafgeyaimk (SEQ ID NO: 551) Capnocytophagamkniqrlgkgnefspfkkedkfyfggflnlannniedffkeiitrfgivitdenkkpketcanimorsus Cc5fgekilneifkkdisivdyekwvnifadyfpftkylslyleemqfknrvicfrdvmkell (NC_015846ktvealrnfythydhepikiedrvfyfldkvildvsltvknkylktdktkeflnqhigee >WP_013997271Alkelckqrkdylvgkgkridkeseiingiynnafkdfickrekqddkenhnsvekilcnkepqnkkqkssatvwelcskssskyteksfpnrendkhclevpisqkgivfllsfflnkgeiyaltsnikgfkakitkeepvtydknsirymathrmfsflaykglkrkirtseinynedgqasstyeketlmlqmldelnkvpdvvyqnlsedvqktfiedwneylkenngdvgtmeeeqvihpvirkryedkfnyfairfldefaqfptlrfqvhlgnylcdkrtkqicdttterevkkkitvfgrlselenkkaiflnereeikgwevfpnpsydfpkenisvnykdfpivgsildrekqpvsnkigirvkiadelgreidkaikekklrnpknrkanqdekqkerlvneivstnsneqgepvvfigqptaylsmndihsvlyeflinkisgealetkivekietqikqiigkdattkilkpytnansnsinrekllrdleqeqqilktlleeqqqrekdkkdkkskrkhelypsekgkvavwlandikrfmpkafkeqwrgyhhsllqkylayyeqskeelknllpkevfkhfpfklkgyfqqqylnqfytdylkrrlsyvnelllniqnfkndkdalkatekecfkffrkqnyiinpiniqiqsilvypiflkrgfldekptmidrekfkenkdteladwfmhyknykednyqkfyayplekveekekfkrnkqinkqkkndvytlmmveyiiqkifgdkfveenplvlkgifqskaerqqnnthaattgerningilnqpkdikiqgkitvkgvklkdignfrkyeidqrvntfldyeprkewmaylpndwkekekqgqlppnnvidrgiskyetvrskillkdvqelekiisdeikeehrhdlkqgkyynfkyyilngllrqlknenvenykvfklntnpekvnitqlkqeatdlegkafvltyirnkfahnqlpkkefwdycqekygkiekektyaeyfaevfkrekealik(SEQ ID NO: 552) Phaeodactylibactermtntpkrrtlhrhpsyfgaflniarhnafmimehlstkydmedkntldeaqlpnaklfgc xiamenensislkkrygkpdvtegvsrdlrryfpflnyplflhlekqqnaeqaatydinpedieftlkgff(NZ_JPOS01000018.1)rllnqmrnnyshyisntdygkfdklpvgdiyeaaifrlldrgkhtkrfdvfeskhtrhle >WP_044218239.1snnseyrprslanspdhentvafvtclflerkyafpflsrldcfrstndaaegdplirkashecytmfccrlpqpklessdilldmvnelgrcpsalynllseedgarfhikreeitgfeedpdeelegeivlkrhsdrfpyfalryfddteafqtlrfdvylgrwrtkpvykkriyggerdrvltqsirtftrlsrllpiyenvkhdavrqneedgklvnpdvtsqfhkswiqiesddraflsdriehfsphynfgdqviglkfinpdryaaiqnvfpklpgeekkdkdaklvnetadaiistheirslflyhylskkpisagderrfiqvdtetfikqyidtiklffediksgelqpiadppnyqkneplpyvrgdkektqeeraqyrerqkeikerrkelntllqnryglsiqyipsrlreyllgykkvpyeklalqklraqrkevkkrikdiekmrtprvgeqatwlaedivfltppkmhtperkttkhpqklnndqfrimqsslayfsvnkkaikkffqketgiglsnretshpflyridvgrcrgildfytgylkykmdwlddaikkvdnrkhgkkeakkyekylpssiqhktpleldytrlpvylprglfkkaivkalaahadfqvepeednvifcldqlldgdtqdfynwqryyrsalteketdnqlvlahpyaegilgtiktlegkqknnklgnkakqkikdelidlkrakrrlldreqylravqaedralwlmiqerqkqkaeheeiafdqldlknitkiltesidarlripdtkvditdklplrrygdlrrvakdrrlvnlasyyhvaglseipydlvkkeleeydrrrvaffehvyqfekevydryaaelrnenpkgestyfshweyvavavkhsadthfnelfkekvmqlrnkfhhnefpyfdwllpevekasaalyadrvfdvaegyyqkmrklmrq (SEQ ID NO: 553)Porphyromonasmntvpasenkgqsrtveddpqyfglylnlarenlieveshvrikfgkkklneeslkqsllgingivalis W83cdhllsvdrwtkvyghsrrylpflhyfdpdsqiekdhdsktgvdpdsaqrlirelyslld(NC_002950.2)flrndfshnrldgttfehlevspdissfitgtyslacgraqsrfadffkpddfvlaknrk >WP_005873511.1eqlisvadgkecltvsglafficlfldreqasgmlsrirgfkrtdenwaravhetfcdlcirhphdrlessntkeallldmlnelnrcprilydmlpeeeraqflpaldensmnnlsenslneesrllwdgssdwaealtkrirhqdrfpylmlrfieemdllkgirfrvdlgeieldsyskkvgrngeydrtitdhalafgklsdfqneeevsrmisgeasypvrfslfapryaiydnkigychtsdpvypksktgekralsnpqsmgfisvhnlrklllmellcegsfsrmqsdflrkanrildetaegklqfsalfpemrhrfippqnpkskdrrekaettlekykqeikgrkdklnsqllsafdmnqrqlpsrlldewmnirpashsvklrtyvkqlnedcrlrlrkfrkdgdgkaraiplvgematflsqdivrmiiseetkklitsayynemqrslaqyageenrrqfraivaelhlldpssghpflsatmetahrytedfykcylekkrewlaktfyrpeqdentkrrisvffvpdgearkllpllirrrmkeqndlqdwirnkqahpidlpshlfdskimellkvkdgkkkwneafkdwwstkypdgmqpfyglrrelnihgksysyipsdgkkfadcythlmektvqdkkrelrtagkpvppdlaadikrsfhravnerefmlrlvqeddrlmlmainkmmtdreedilpglknidsildeenqfslavhakvlekegeggdnslslvpatieikskrkdwskyiryrydrrvpglmshfpehkatldevktllgeydrcrikifdwafalegaimsdrdlkpylhesssregksgehstivkmlvekkgcltpdesqylilirnkaahnqfpcaaempliyrdvsakvgsiegssakdlpegsslvdslwkkyemiirkilpildpenrffgkllnnmsqpindl (SEQ ID NO: 554)Porphyromonasmntvpasenkgqsrtveddpqyfglylnlarenlieveshvrikfgkkklneeslkqsllgingivalis F0570cdhllsvdrwtkvyghsrrylpflhyfdpdsqiekdhdsktgvdpdsaqrlirelyslld(NZ_K1259168.1)flrndfshnrldgttfehlevspdissfitgtyslacgraqsrfadffkpddfvlaknrk >WP_021665475.1eqlisvadgkecltvsglafficlfldreqasgmlsrirgfkrtnenwaravhetfcdlcirhphdrlessntkeallldmlnelnrcprilydmlpeeeraqflpaldensmnnlsenslneesrllwdgssdwaealtkrirhqdrfpylmlrfieemdllkgirfrvdlgeieldsyskkvgrngeydrtitdhalafgklsdfqneeevsrmisgeasypvrfslfapryaiydnkigychtsdpvypksktgekralsnpqsmgfisvhdlrklllmellcegsfsrmqsgflrkanrildetaegklqfsalfpemrhrfippqnpkskdrrekaettlekykqeikgrkdklnsqllsafdmnqrqlpsrlldewmnirpashsvklrtyvkqlnedcrlrlrkfrkdgdgkaraiplvgematflsqdivrmiiseetkklitsayynemqrslaqyageenrrqfraivaelhlldpssghpflsatmetahrytedfykcylekkrewlaktfyrpeqdentkrrisvffvpdgearkllpllirrrmkeqndlqdwirnkqahpidlpshlfdskimellkvkdgkkkwneafkdwwstkypdgmqpfyglrrelnihgksysyipsdgkkfadcythlmektvqdkkrelrtagkpvppdlaadikrsfhravnerefmlrlvqeddrlmlmainkmmtdreedilpglknidsildkenqfslavhakvlekegeggdnslslvpatieikskrkdwskyiryrydrrvpglmshfpehkatldevktllgeydrcrikifdwafalegaimsdrdlkpylhesssregksgehstivkmlvekkgcltpdesqylilirnkaahnqfpcaaempliyrdvsakvgsiegssakdlpegsslvdslwkkyemiirkilpildhenrffgkllnnmsqpindl (SEQ ID NO: 555)Porphyromonasmntvpasenkgqsrtveddpqyfglylnlarenlieveshvrikfgkkklneeslkqsll gingivaliscdhllsvdrwtkvyghsrrylpflhyfdpdsqiekdhdsktgvdpdsaqrlirelyslld ATCC 33277flrndfshnrldgttfehlevspdissfitgtyslacgraqsrfavffkpddfvlaknrk(NC 010729.1)eqlisvadgkecltvsgfafficlfldreqasgmlsrirgfkrtdenwaravhetfcdlc >NV_P012458151.1irhphdrlessntkeallldmlnelnrcprilydmlpeeeraqflpaldensmnnlsensldeesrllwdgssdwaealtkrirhqdrfpylmlrfieemdllkgirfrvdlgeieldsyskkvgrngeydrtitdhalafgklsdfqneeevsrmisgeasypvrfslfapryaiydnkigychtsdpvypksktgekralsnpqsmgfisvhdlrklllmellcegsfsrmqsdflrkanrildetaegklqfsalfpemrhrfippqnpkskdrrekaettlekykqeikgrkdklnsqllsafdmdqrqlpsrlldewmnirpashsvklrtyvkqlnedcrlrlrkfrkdgdgkaraiplvgematflsqdivrmiiseetkklitsayynemqrslaqyageenrrqfraivaelrlldpssghpflsatmetahrytegfykcylekkrewlakifyrpeqdentkrrisvffvpdgearkllpllirrrmkeqndlqdwirnkqahpidlpshlfdskvmellkvkdgkkkwneafkdwwstkypdgmqpfyglrrelnihgksysyipsdgkkfadcythlmektvrdkkrelrtagkpvppdlaadikrsfhravnerefmlrlvqeddrlmlmainkmmtdreedilpglknidsildeenqfslavhakvlekegeggdnslslvpatieikskrkdwskyiryrydrrvpglmshfpehkatldevktllgeydrcrikifdwafalegaimsdrdlkpylhesssregksgehstivkmlvekkgcltpdesqylilirnkaahnqfpcaaempliyrdvsakvgsiegssakdlpegsslvdslwkkyemiirkilpildpenrffgkllnnmsqpindl (SEQ ID NO: 556)Porphyromonasmntvpasenkgqsrtveddpqyfglylnlarenlieveshvrikfgkkklneeslkqsllgingivalis F0185cdhllsvdrwtkvyghsrrylpflhyfdpdsqiekdhdsktgvdpdsaqrlirelyslld(AWVC01000122.1)flrndfshnrldgttfehlevspdissfitgtyslacgraqsrfadffkpddfvlaknrk >ERJ81987.1eqlisvadgkecltvsglafficlfldreqasgmlsrirgfkrtdenwaravhetfcdlcirhphdrlessntkeallldmlnelnrcprilydmlpeeeraqflpaldensmnnlsenslneesrllwdgssdwaealtkrirhqdrfpylmlrfieemdllkgirfrvdlgeieldsyskkvgrngeydrtitdhalafgklsdfqneeevsrmisgeasypvrfslfapryaiydnkigychtsdpvypksktgekralsnpqsmgfisvhdlrklllmellcegsfsrmqsgflrkanrildetaegklqfsalfpemrhrfippqnpkskdrrekaettlekykqeikgrkdklnsqllsafdmnqrqlpsrlldewmnirpashsvklrtyvkqlnedcrlrlrkfrkdgdgkaraiplvgematflsqdivrmiiseetkklitsayynemqrslaqyageenrrqfraivaelhlldpssghpflsatmetahrytedfykcylekkrewlaktfyrpeqdentkrrisvffvpdgearkllpllirrrmkeqndlqdwirnkqahpidlpshlfdskimellkvkdgkkkwneafkdwwstkypdgmqpfyglrrelnihgksysyipsdgkkfadcythlmektvqdkkrelrtagkpvppdlaadikrsfhravnerefmlrlvqeddrlmlmainkmmtdreedilpglknidsildeenqfslavhakvlekegeggdnslslvpatieikskrkdwskyiryrydrrvpglmshfpehkatldevktllgeydrcrikifdwafalegaimsdrdlkpylhesssregksgehstivkmlvekkgcltpdesqylilirnkaahnqfpcaaempliyrdvsakvgsiegssakdlpegsslvdslwkkyemiirkilpildhenrffgkllnnmsqpindl (SEQ ID NO: 557)Porphyromonasmntvpasenkgqsrtveddpqyfglylnlarenlieveshvrikfgkkklneeslkqsllgingivalis F0185cdhllsvdrwtkvyghsrrylpflhyfdpdsqiekdhdsktgvdpdsaqrlirelyslld(NZ_K1259960.1)flrndfshnrldgttfehlevspdissfitgtyslacgraqsrfadffkpddfvlaknrk >WP_021677657.1eqlisvadgkecltvsglafficlfldreqasgmlsrirgfkrtdenwaravhetfcdlcirhphdrlessntkeallldmlnelnrcprilydmlpeeeraqflpaldensmnnlsenslneesrllwdgssdwaealtkrirhqdrfpylmlrfieemdllkgirfrvdlgeieldsyskkvgrngeydrtitdhalafgklsdfqneeevsrmisgeasypvrfslfapryaiydnkigychtsdpvypksktgekralsnpqsmgfisvhdlrklllmellcegsfsrmqsgflrkanrildetaegklqfsalfpemrhrfippqnpkskdrrekaettlekykqeikgrkdklnsqllsafdmnqrqlpsrlldewmnirpashsvklrtyvkqlnedcrlrlrkfrkdgdgkaraiplvgematflsqdivrmiiseetkklitsayynemqrslaqyageenrrqfraivaelhlldpssghpflsatmetahrytedfykcylekkrewlaktfyrpeqdentkrrisvffvpdgearkllpllirrrmkeqndlqdwirnkqahpidlpshlfdskimellkvkdgkkkwneafkdwwstkypdgmqpfyglrrelnihgksysyipsdgkkfadcythlmektvqdkkrelrtagkpvppdlaadikrsfhravnerefmlrlvqeddrlmlmainkmmtdreedilpglknidsildeenqfslavhakvlekegeggdnslslvpatieikskrkdwskyiryrydrrvpglmshfpehkatldevktllgeydrcrikifdwafalegaimsdrdlkpylhesssregksgehstivkmlvekkgcltpdesqylilirnkaahnqfpcaaempliyrdvsakvgsiegssakdlpegsslvdslwkkyemiirkilpildhenrffgkllnnmsqpindl (SEQ ID NO: 558)Porphyromonasmntvpasenkgqsrtveddpqyfglylnlarenlieveshvrikfgkkklneeslkqsllgingivalis SJD2cdhllsvdrwtkvyghsrrylpflhyfdpdsqiekdhdsktgvdpdsaqrlirelyslld(NZ_K1629875.1)flrndfshnrldgttfehlevspdissfitgtyslacgraqsrfadffkpddfvlaknrk >WP_023846767.1eqlisvadgkecltvsglafficlfldreqasgmlsrirgfkrtdenwaravhetfcdlcirhphdrlessntkeallldmlnelnrcprilydmlpeeeraqflpaldensmnnlsenslneesrllwdgssdwaealtkrirhqdrfpylmlrfieemdllkgirfrvdlgeieldsyskkvgrngeydrtitdhalafgklsdfqneeevsrmisgeasypvrfslfapryaiydnkigychtsdpvypksktgekralsnprsmgfisvhdlrklllmellcegsfsrmqsdflrkanrildetaegklqfsalfpemrhrfippqnpkskdrrekaettlekykqeikgrkdklnsqllsafdmnqrqlpsrlldewmnirpashsvklrtyvkqlnedcrlrlrkfrkdgdgkaraiplvgematflsqdivrmiiseetkklitsayynemqrslaqyageenrrqfraivaelhlldpssghpflsatmetahrytedfykcylekkrewlaktfyrpeqdentkrrisvffvpdgearkllpllirrrmkeqndlqdwirnkqahpidlpshlfdskimellkvkdgkkkwneafkdwwstkypdgmqpfyglrrelnihgksysyipsdgkkfadcythlmektvqdkkrelrtagkpvppdlaadikrsfhravnerefmlrlvqeddrlmlmainkmmtdreedilpglknidsildeenqfslavhakvlekegeggdnslslvpatieikskrkdwskyiryrydrrvpglmshfpehkatldevktllgeydrcrikifdwafalegaimsdrdlkpylhesssregksgehstivkmlvekkgcltpdesqylilirnkaahnqfpcaaempliyrdvsakvgsiegssakdlpegsslvdslwkkyemiirkilpildpenrffgkllnnmsqpindl (SEQ ID NO: 559)Porphyromonasmntvpasenkgqsrtveddpqyfglylnlarenlieveshvrikfgkkklneeslkqsllgingivalis F0568cdhllsvdrwtkvyghsrrylpflhyfdpdsqiekdhdsktgvdpdsaqrlirelyslld(AWUU01000145.1)flrndfshnrldgttfehlevspdissfitgtyslacgraqsrfadffkpddfvlaknrk >ERJ65637.1eqlisvadgkecltvsglafficlfldreqasgmlsrirgfkrtdenwaravhetfcdlcirhphdrlessntkeallldmlnelnrcprilydmlpeeeraqflpaldensmnnlsenslneesrllwdgssdwaealtkrirhqdrfpylmlrfieemdllkgirfrvdlgeieldsyskkvgrngeydrtitdhalafgklsdfqneeevsrmisgeasypvrfslfapryaiydnkigychtsdpvypksktgekralsnprsmgfisvhdlrklllmellcegsfsrmqsdflrkanrildetaegklqfsalfpemrhrfippqnpkskdrrekaettlekykqeikgrkdklnsqllsafdmdqrqlpsrlldewmnirpashsvklrtyvkqlnedcrlrlqkfrkdgdgkaraiplvgematflsqdivrmiiseetkklitsayynemqrslaqyageenrhqfraivaelrlldpssghpflsatmetahrytedfykcylekkrewlaktfyrpeqdentkrrisvffvpdgearkllpllirrrmkeqndlqdwirnkqahpidlpshlfdskimellkvkdgkkkwneafkdwwstkypdgmqpfyglrrelnihgksysyipsdgkkfadcythlmektvqdkkrelrtagkpvppdlaadikrsfhravnerefmlrlvqeddrlmlmainkmmtdreedilpglknidsildeenqfslavhakvlekegeggdnslslvpatieikskrkdwskyiryrydrrvpglmshfpehkatldevktllgeydrcrikifdwafalegaimsdrdlkpylhesssregksgehstivkmlvekkgcltpdesqylilirnkaahnqfpcaaempliyrdvsakvgsiegssakdlpegsslvdslwkkyemiirkilpildpenrffgkllnnmsqpindl (SEQ ID NO: 560)Porphyromonasmntvpasenkgqsrtveddpqyfglylnlarenlieveshvrikfgkkklneeslkqsllgingivalis W4087cdhllsvdrwtkvyghsrrylpflhyfdpdsqiekdhdsktgvdpdsaqrlirelyslld(AWVE01000130.1)flrndfshnrldgttfehlevspdissfitgtyslacgraqsrfadffkpddfvlaknrk >ERJ87335.1eqlisvadgkecltvsglafficlfldreqasgmlsrirgfkrtdenwaravhetfcdlcirhphdrlessntkeallldmlnelnrcprilydmlpeeeraqflpaldensmnnlsenslneesrllwdgssdwaealtkrirhqdrfpylmlrfieemdllkgirfrvdlgeieldsyskkvgrngeydrtitdhalafgklsdfqneeevsrmisgeasypvrfslfapryaiydnkigychtsdpvypksktgekralsnprsmgfisvhdlrklllmellcegsfsrmqsdflrkanrildetaegklqfsalfpemrhrfippqnpkskdrrekaettlekykqeikgrkdklnsqllsafdmdqrqlpsrlldewmnirpashsvklrtyvkqlnedcrlrlqkfrkdgdgkaraiplvgematflsqdivrmiiseetkklitsayynemqrslaqyageenrhqfraivaelrlldpssghpflsatmetahrytedfykcylekkrewlaktfyrpeqdentkrrisvffvpdgearkllpllirrrmkeqndlqdwirnkqahpidlpshlfdskvmellkvkdgkkkwneafkdwwstkypdgmqpfyglrrelnihgksysyipsdgkkfadcythlmektvrdkkrelrtagkpvppdlaayikrsfhravnerefmlrlvqeddrlmlmainkimtdreedilpglknidsildkenqfslavhakvlekegeggdnslslvpatieikskrkdwskyiryrydrrvpglmshfpehkatldevktllgeydrcrikifdwafalegaimsdrdlkpylhesssregksgehstivkmlvekkgcltpdesqylilirnkaahnqfpcaaeipliyrdvsakvgsiegssakdlpegsslvdslwkkyemiirkilpildpenrffgkllnnmsqpindl (SEQ ID NO: 561)Porphyromonasmntvpasenkgqsrtveddpqyfglylnlarenlieveshvrikfgkkklneeslkqsllgingivalis W4087cdhllsvdrwtkvyghsrrylpflhyfdpdsqiekdhdsktgvdpdsaqrlirelyslld(NZ_K1260263.1)flrndfshnrldgttfehlevspdissfitgtyslacgraqsrfadffkpddfvlaknrk >WP_021680012.1eqlisvadgkecltvsglafficlfldreqasgmlsrirgfkrtdenwaravhetfcdlcirhphdrlessntkeallldmlnelnrcprilydmlpeeeraqflpaldensmnnlsenslneesrllwdgssdwaealtkrirhqdrfpylmlrfieemdllkgirfrvdlgeieldsyskkvgrngeydrtitdhalafgklsdfqneeevsrmisgeasypvrfslfapryaiydnkigychtsdpvypksktgekralsnprsmgfisvhdlrklllmellcegsfsrmqsdflrkanrildetaegklqfsalfpemrhrfippqnpkskdrrekaettlekykqeikgrkdklnsqllsafdmdqrqlpsrlldewmnirpashsvklrtyvkqlnedcrlrlqkfrkdgdgkaraiplvgematflsqdivrmiiseetkklitsayynemqrslaqyageenrhqfraivaelrlldpssghpflsatmetahrytedfykcylekkrewlaktfyrpeqdentkrrisvffvpdgearkllpllirrrmkeqndlqdwirnkqahpidlpshlfdskvmellkvkdgkkkwneafkdwwstkypdgmqpfyglrrelnihgksysyipsdgkkfadcythlmektvrdkkrelrtagkpvppdlaayikrsfhravnerefmlrlvqeddrlmlmainkimtdreedilpglknidsildkenqfslavhakvlekegeggdnslslvpatieikskrkdwskyiryrydrrvpglmshfpehkatldevktllgeydrcrikifdwafalegaimsdrdlkpylhesssregksgehstivkmlvekkgcltpdesqylilirnkaahnqfpcaaeipliyrdvsakvgsiegssakdlpegsslvdslwkkyemiirkilpildpenrffgkllnnmsqpindl (SEQ ID NO: 562)Porphyromonasmntvpasenkgqsrtveddpqyfglylnlarenlieveshvrikfgkkklneeslkqsllgingivalis F0568cdhllsvdrwtkvyghsrrylpflhyfdpdsqiekdhdsktgvdpdsaqrlirelyslld(NZ_K1258981.1)flrndfshnrldgttfehlevspdissfitgtyslacgraqsrfadffkpddfvlaknrk >WP_021663197.1eqlisvadgkecltvsglafficlfldreqasgmlsrirgfkrtdenwaravhetfcdlcirhphdrlessntkeallldmlnelnrcprilydmlpeeeraqflpaldensmnnlsenslneesrllwdgssdwaealtkrirhqdrfpylmlrfieemdllkgirfrvdlgeieldsyskkvgrngeydrtitdhalafgklsdfqneeevsrmisgeasypvrfslfapryaiydnkigychtsdpvypksktgekralsnprsmgfisvhdlrklllmellcegsfsrmqsdflrkanrildetaegklqfsalfpemrhrfippqnpkskdrrekaettlekykqeikgrkdklnsqllsafdmdqrqlpsrlldewmnirpashsvklrtyvkqlnedcrlrlqkfrkdgdgkaraiplvgematflsqdivrmiiseetkklitsayynemqrslaqyageenrhqfraivaelrlldpssghpflsatmetahrytedfykcylekkrewlaktfyrpeqdentkrrisvffvpdgearkllpllirrrmkeqndlqdwirnkqahpidlpshlfdskimellkvkdgkkkwneafkdwwstkypdgmqpfyglrrelnihgksysyipsdgkkfadcythlmektvqdkkrelrtagkpvppdlaadikrsfhravnerefmlrlvqeddrlmlmainkmmtdreedilpglknidsildeenqfslavhakvlekegeggdnslslvpatieikskrkdwskyiryrydrrvpglmshfpehkatldevktllgeydrcrikifdwafalegaimsdrdlkpylhesssregksgehstivkmlvekkgcltpdesqylilirnkaahnqfpcaaempliyrdvsakvgsiegssakdlpegsslvdslwkkyemiirkilpildpenrffgkllnnmsqpindl (SEQ ID NO: 563)Porphyromonasmntvpasenkgqsrtveddpqyfglylnlarenlieveshvrikfgkkklneeslkqsll gingivaliscdhllsvdrwtkvyghsrrylpflhyfdpdsqiekdhdsktgvdpdsaqrlirelyslld(NZ_LOEL01000010.1)flrndfshnrldgttfehlevspdissfitgtyslacgraqsrfadffkpddfvlaknrk >WP_061156637.1eqlisvadgkecltvsglafficlfldreqasgmlsrirgfkrtdenwaravhetfcdlcirhphdrlessntkeallldmlnelnrcprilydmlpeeeraqflpaldensmnnlsenslneesrllwdgssdwaealtkrirhqdrfpylmlrfieemdllkgirfrvdlgeieldsyskkvgrngeydrtitdhalafgklsdfqneeevsrmisgeasypvrfslfapryaiydnkigychtsdpvypksktgekralsnpqsmgfisvhdlrklllmellcegsfsrmqsgflrkanrildetaegklqfsalfpemrhrfippqnpkskdrrekaettlekykqeikgrkdklnsqllsafdmnqrqlpsrlldewmnirpashsvklrtyvkqlnedcrlrlrkfrkdgdgkaraiplvgematflsqdivrmiiseetkklitsayynemqrslaqyageenrrqfraivaelhlldpssghpflsatmetahrytedfykcylekkrewlaktfyrpeqdentkrrisvffvpdgearkllpllirrrmkeqndlqdwirnkqahpidlpshlfdskimellkvkdgkkkwneafkdwwstkypdgmqpfyglrrelnihgksysyipsdgkkfadcythlmektvqdkkrelrtagkpvppdlaadikrsfhravnerefmlrlvqeddrlmlmainkmmtdreedilpglknidsildkenqfslavhakvlekegeggdnslslvpatieikskrkdwskyiryrydrrvpglmshfpehkatldevktllgeydrcrikifdwafalegaimsdrdlkpylhesssregksgehstivkmlvekkgcltpdesqylilirnkaahnqfpcaaempliyrdvsakvgsiegssakdlpegsslvdslwkkyemiirkilpildpenrffgkllnnmsqpindl (SEQ ID NO: 564)Porphyromonasmntvpatenkgqsrtveddpqyfglylnlarenlieveshvrikfgkkklneeslkqsll gulaecdhllsidrwtkvyghsrrylpflhcfdpdsgiekdhdsktgvdpdsaqrlirelyslld(NZ_JRAQ01000019.1)flrndfshnrldgttfehlkvspdissfitgaytfaceraqsrfadffkpddfllaknrk >NV1_039445055.1eqlisvadgkecltvsgfafficlfldreqasgmlsrirgfkrtdenwaravhetfcdlcirhphdrlessntkeallldmlnelnrcprilydmlpeeeraqflpaldensmnnlsenslneesrllwdgssdwaealtkrirhqdrfpylmlrfieemdllkgirfrvdlgeieldsyskkvgrngeydrtitdhalafgklsdfqneeevsrmisgeasypvrfslfapryaiydnkigychtsdpvypksktgekralsnpqsmgfisvhdlrklllmellcegsfsrmqsdflrkanrildetaegklqfsalfpemrhrfippqnpkskdrrekaettlekykqeikgrkdklnsqllsafdmnqrqlpsrlldewmnirpashsvklrtyvkqlnedcrlrlrkfrkdgdgkaraiplvgematflsqdivrmiiseetkklitsayynemqrslaqyageenrrqfraivaelhlldpssghpflsatmetahrytedfykcylekkrewlaktfyrpeqdentkrrisvffvpdgearkllpllirrrmkeqndlqdwirnkqahpidlpshlfdskimellkvkdgkkkwneafkdwwstkypdgmqpfyglrrelnihgksysyipsdgkkfadcythlmektvrdkkrelrtagkpvppdlaayikrsfhravnerefmlrlvqeddrlmlmainkmmtdreedilpglknidsildeenqfslavhakvlekegeggdnslslvpatieikskrkdwskyiryrydrrvpglmshfpehkatldevktllgeydrcrikifdwafalegaimsdrdlkpylhesssregksgehstivkmlvekkgcltpdesqylilirnkaahnqfpcaaempliyrdvsakvgsiegssakdlpegsslvdslwkkyemiirkilpildhenrffgkllnnmsqpindl (SEQ ID NO: 565)Bacteroides mesiknsqkstgktlqkdppyfglylnmallnvrkvenhirkwlgdvallpeksgfhsllpyogenes ttdnlssakwtrfyyksrkflpflemfdsdkksyenrrettecldtidrqkissllkevyF0041 gklqdirnafshyhiddqsvkhtaliissemhrfienaysfalqktrarftgvfvetdfl(KE993153.1)qaeekgdnkkffaiggnegiklkdnalifliclfldreeafkflsratgfkstkekgfla >ER181700.1vretfcalccrqpherllsvnpreallmdmlnelnrcpdilfemldekdqksflpllgeeeqahilenslndelceaiddpfemiaslskrvryknrfpylmlryieeknllpfirfridlgclelasypkkmgeennyersvtdhamafgrltdfhnedavlqqitkgitdevrfslyapryaiynnkigfvrtggsdkisfptlkkkggeghcvaytlqntksfgfisiydlrkilllsfldkdkaknivsglleqcekhwkdlsenlfdairtelqkefpvplirytlprskggklvsskladkqekyeseferrkeklteilsekdfdlsqiprrmidewlnvlptsrekklkgyvetlkldcrerlrvfekrekgehpvpprigematdlakdiirmvidqgvkqritsayyseiqrclaqyagddnrrhldsiirelrlkdtknghpflgkvlrpglghteklyqryfeekkewleatfypaaspkrvprfvnpptgkqkelpliirnlmkerpewrdwkqrknshpidlpsqlfeneicrllkdkigkepsgklkwnemfklywdkefpngmqrfyrckrrvevfdkvveyeyseeggnykkyyealidevvrqkissskeksklqvedltlsvrrvfkrainekeyqlrllceddrllfmavrdlydwkeaqldldkidnmlgepvsysqviqleggqpdavikaecklkdvsklmrycydgrvkglmpyfanheatqeqvemelrhyedhrrrvfnwvfaleksvlkneklrrfyeesqggcehrrcidalrkaslvseeeyeflvhirnksahnqfpdleigklppnvtsgfceciwskykaiicriipfidperrffgklleqk (SEQ ID NO: 566) Bacteroidesmesiknsqkstgktlqkdppyfglylnmallnvrkvenhirkwlgdvallpeksgfhsll pyogenesttdnlssakwtrfyyksrkflpflemfdsdkksyenrretaecldtidrqkissllkevy JCM 10003gklqdirnafshyhiddqsvkhtaliissemhrfienaysfalqktrarftgvfvetdfl(NZ_BAIU01000001.1)qaeekgdnkkffaiggnegiklkdnalifliclfldreeafkflsratgfkstkekgfla >WP_034542281.1vretfcalccrqpherllsvnpreallmdmlnelnrcpdilfemldekdqksflpllgeeeqahilenslndelceaiddpfemiaslskrvryknrfpylmlryieeknllpfirfridlgclelasypkkmgeennyersvtdhamafgrltdfhnedavlqqitkgitdevrfslyapryaiynnkigfvrtsgsdkisfptlkkkggeghcvaytlqntksfgfisiydlrkilllsfldkdkaknivsglleqcekhwkdlsenlfdairtelqkefpvplirytlprskggklvsskladkqekyeseferrkeklteilsekdfdlsqiprrmidewlnvlptsrekklkgyvetlkldcrerlrvfekrekgehplpprigematdlakdiirmvidqgvkqritsayyseiqrclaqyagddnrrhldsiirelrlkdtknghpflgkvlrpglghteklyqryfeekkewleatfypaaspkrvprfvnpptgkqkelpliirnlmkerpewrdwkqrknshpidlpsqlfeneicrllkdkigkepsgklkwnemfklywdkefpngmqrfyrckrrvevfdkvveyeyseeggnykkyyealidevvrqkissskeksklqvedltlsvrrvfkrainekeyqlrllceddrllfmavrdlydwkeaqldldkidnmlgepvsysqviqleggqpdavikaecklkdvsklmrycydgrvkglmpyfanheatqeqvemelrhyedhrrrvfnwvfaleksvlkneklrrfyeesqggcehrrcidalrkaslvseeeyeflvhirnksahnqfpdleigklppnvtsgfceciwskykaiicriipfidperrffgklleqk (SEQ ID NO: 567) Alistipesmsneigafrehqfayapgnekqeeatfatyfnlalsnvegmmfgevesnpdkieksldtl sp. ZOR0009ppailrgiasfiwlskedhpdkaysteevkvivtdlvrrlcfyrnyfshcfyldtqyfys(NZ_JTLD01000029.1)delvdttaigeklpynfhhfitnrlfryslpeitlfrwnegerkyeilrdgliffcclfl >WP_047447901.1krgqaerflnelrffkrtdeegrikrtiftkyctreshkhigieeqdflifqdiigdlnrvpkvcdgvvdlskeneryiknretsnesdenkaryrllirekdkfpyylmryivdfgvlpcitfkqndystkegrgqfhpadaavageercynfvvrngnvyysympqaqnvvriselqgtisveelrnmvyasingkdvnksveqylyhlhllyekiltisgqtikegrvdvedyrplldklllrpasngeelrrelrkllpkrvcdllsnrfdcsegvsavekrlkaillrheqlllsqnpalhidkiksvidylylffsddekfrqqptekahrglkdeefqmyhylvgdydshplalwkeleasgrlkpemrkltsatslhglymlclkgtvewcrkqlmsigkgtakveaiadryglklydklkeytpeqlerevklvvmhgyaaaatpkpkaqaaipskltelrfysflgkremsfaafirqdkkaqklwlrnfytveniktlqkrqaaadaackklynlvgevervhtndkvlvlvaqryrerllnvgskcavtldnperqqkladvyevqnawlsirfddldftlthvnlsnlrkaynliprkhilafkeyldnrvkqklceecrnvrrkedlctccsprysnitswlkenhsessiereaatmmlldverkllsfllderrkaiieygkfipfsalvkecrladaglcgirndvlhdnvisyadaigklsayfpkeaseaveyirrtkevreqrreelmanssq (SEQ ID NO: 568)Flavobacteriummenlnkildkeneiciskifntkgiaapitekaldnikskqkndlnkearlhyfsighsfbranchiophilumkqidtkkvfdyvlieelkdekplkfitlqkdfftkefsiklqklinsirninnhyvhnfn FL-15dinlnkidsnvfhflkesfelaiiekyykvnkkypldneivlflkelfikdentallnyf(NC_016001.1)tnlskdeaieyiltftitenkiwninnehnilniekgkyltfeamlflitiflykneanh >WP_014084666.1llpklydfknnkskqelftffskkftsqdidaeeghlikfrdmiqylnhyptawnndlklesenknkimttklidsiiefelnsnypsfatdiqfkkeakaflfasnkkrnqtsfsnksyneeirhnphikqyrdeiasaltpisfnvkedkfkifvkkhvleeyfpnsigyekfleyndftekekedfglklysnpktnklieridnhklvkshgrnqdrfmdfsmrflaennyfgkdaffkcykfydtqeqdeflqsnennddvkfhkgkvttyikyeehlknysywdcpfveennsmsvkisigseekilkiqrnlmiyflenalynenvenqgyklvnnyyrelkkdveesiasldliksnpdfkskykkilpkrllhnyapakqdkapenafetllkkadfreeqykkllkkaeheknkedfvkrnkgkqfklhfirkacqmmyfkekyntlkegnaafekkdpviekrknkehefghhknlnitreefndyckwmfafngndsykkylrdlfsekhffdnqeyknlfessvnleafyaktkelfkkwietnkptnnenrytlenyknlilqkqvfinvyhfskylidknllnsennviqykslenveylisdfyfqsklsidqyktcgklfnklksnkledcllyeiaynyidkknvhkidigkiltskiiltindantpykisvpfnklerytemiaiknqnnlkarflidlplylsknkikkgkdsagyeiiikndleiedintinnkiindsvkftevlmelekyfilkdkcilsknyidnseipslkqfskvwikeneneiinyrniachfhlplletfdnlllnveqkfikeelqnvstindlskpqeylillfikfkhnnfylnlfnknesktikndkevkknrvlqkfinqvilkkk (SEQ ID NO: 569) Prevotellamskeckkgrqekkrrlqkanfsisltgkhvfgayfnmartnfvktinyilpiagvrgnys sp. MA2016enqinkmlhalfligagrneeltteqkqwekklrinpeqqtkfqkllfkhfpvlgpmmad(NZ_JHUW01000010.1)vadhkaylnkkkstvqtedetfamlkgvsladcldiiclmadtltecrnfythkdpynkp >WP_036929175.1sqladqylhqemiakkldkvvvasrrilkdreglsvnevefltgidhlhqevlkdefgnakvkdgkvmktfveyddfyfkisgkrlvngytvttkddkpvnvntmlpalsdfgllyfcvlflskpyaklfidevrlfeyspfddkenmimsemlsiyrirtprlhkidshdskatlamdifgelrrcpmelynlldknagqpffhdevkhpnshtpdvskrlryddrfptlalryidetelfkrirfqlqlgsfrykfydkencidgrvrvrriqkeingygrmqevadkrmdkwgdliqkreersvkleheelyinldqfledtadstpyvtdrrpaynihanriglywedsqnpkqykvfdengmyipelvvtedkkapikmpaprcalsvydlpamlfyeylreqqdnefpsaeqviieyeddyrkffkavaegklkpfkrpkefrdflkkeypklrmadipkklqlflcshglcynnkpetvyerldrltlqhleerelhiqnrlehyqkdrdmignkdnqygkksfsdvrhgalarylaqsmmewqptklkdkekghdkltglnynvltaylatyghpqvpeegftprtleqvlinahliggsnphpfinkvlalgnrnieelylhyleeelkhirsriqslssnpsdkalsalpfihhdrmryhertseemmalaaryttiqlpdglftpyileilqkhytensdlqnalsqdvpvklnptcnaaylitlfyqtvlkdnaqpfylsdktytrnkdgekaesfsfkrayelfsvlnnnkkdtfpfemiplfltsdeigerlsaklldgdgnpvpevgekgkpatdsqgntiwkrriysevddyaekltdrdmkisfkgeweklprwkqdkiikrrdetrrqmrdellqrmpryirdikdnertlrryktqdmvlfllaekmftniiseqssefnwkqmrlskvcneaflrqtltfrvpvtvgettiyvegenmslknygefyrfltddrlmsllnnivetlkpnengdlvirhtdlmselaaydqyrstifmliqsienliitnnavlddpdadgfwvredlpkrnnfasllelinqlnnveltdderkllvairnafshnsynidfslikdvkhlpevakgilqhlqsmlgveitk (SEQ ID NO: 570) Myroidesmkdilttdttekqnrfyshkiadkyffggyfnlasnniyevfeevnkrntfgklakrdng odomtimimusnlknyiihvfkdelsisdfekrvaifasyfpiletvdkksikernrtidltlsgrirqfr CCUG 10230emlislvtavdqlrnfythyhhsdivienkvldflnssfvstalhvkdkylktdktkefl(AGEC02000017.1)ketiaaeldilieaykkkqiekkntrfkankredilnaiyneafwsfindkdkdkdketv >EHO06562.1vakgadayfeknhhksndpdfalnisekgivyllsffltnkemdslkanitgfkgkvdresgnsikymatqrlysfhtyrglkqkirtseegvketllmqmidelskvpnvvyqhlsttqqnsfiedwneyykdyeddvetddlsrvihpvirkryedrfnyfairfldeffdfptlrfqvhlgdyvhdrrtkqlgkvesdriikekvtvfarlkdinsakasyfhsleeqdkeeldnkwtlfpnpsydfpkehtlqhqgeqknagkigiyvklrdtqykekaaleearkslnpkersatkaskydiitqiieandnvksekplvftgqpiaylsmndihsmlfslltdnaelkktpeeveaklidgigkqineilskdtdtkilkkykdndlketdtdkitrdlardkeeieklileqkqraddynytsstkfnidksrkrkhllfnaekgkigvwlandikrfmfkeskskwkgyqhtelqklfayfdtsksdlelilsnmvmvkdypielidlvkksrtivdflnkylearleyienvitrvknsigtpqfktvrkecftflkksnytvvsldkqverilsmplfiergfmddkptmlegksykqhkekfadwfvhykensnyqnfydtevyeittedkrekakvtkkikqqqkndvftlmmvnymleevlklssndrlslnelyqtkeerivnkqvakdtgernknyiwnkvvdlqlcdglvhidnvklkdignfrkyendsrvkefltyqsdivwsaylsnevdsnklyvierqldnyesirskellkevqeiecsvynqvankeslkqsgnenfkqyvlqgllpigmdvremlilstdvkfkkeeiiqlgqageveqdlysliyirnkfahnqlpikeffdfcennyrsisdneyyaeyymeifrsikekyan (SEQ ID NO: 571) Myroidesmkdilttdttekqnrfyshkiadkyffggyfnlasnniyevfeevnkrntfgklakrdng odomtimimusnlknyiihvfkdelsisdfekrvaifasyfpiletvdkksikernrtidltlsgrirqfr CCUG 3837emlislvtavdqlrnfythyhhseivienkvldflnsslvstalhvkdkylktdktkefl(AGEC01000016.1)ketiaaeldilieaykkkqiekkntrfkankredilnaiyneafwsfindkdkdketvva >EKB06014.1kgadayfeknhhksndpdfalnisekgivyllsffltnkemdslkanitgfkgkvdresgnsikymatqrlysfhtyrglkqkirtseegvketllmqmidelskvpnvvyqhlsttqqnsfiedwneyykdyeddvetddlsrvihpvirkryedrfnyfairfldeffdfptlrfqvhlgdyvhdrrtkqlgkvesdriikekvtvfarlkdinsakasyfhsleeqdkeeldnkwtlfpnpsydfpkehtlqhqgeqknagkigiyvklrdtqykekaaleearkslnpkersatkaskydiitqiieandnvksekplvftgqpiaylsmndihsmlfslltdnaelkktpeeveaklidgigkqineilskdtdtkilkkykdndlketdtdkitrdlardkeeieklileqkqraddynytsstkfnidksrkrkhllfnaekgkigvwlandikrfmfkeskskwkgyqhtelqklfayfdtsksdlelilsdmvmvkdypielidlvrksrtivdflnkylearlgyienvitrvknsigtpqfktvrkecfaflkesnytvasldkqierilsmplfiergfmdskptmlegksyqqhkedfadwfvhykensnyqnfydtevyeiitedkreqakvtkkikqqqkndvftlmmvnymleevlklpsndrlslnelyqtkeerivnkqvakdtgernknyiwnkvvdlqlceglvridkvklkdignfrkyendsrvkefltyqsdivwsgylsnevdsnklyvierqldnyesirskellkevqeiecivynqvankeslkqsgnenfkqyvlqgllprgtdvremlilstdvkfkkeeimqlgqvreveqdlysliyirnkfahnqlpikeffdfcennyrpisdneyyaeyymeifrsikekyas (SEQ ID NO: 572) Myroidesmkdilttdttekqnrfyshkiadkyffggyfnlasnniyevfeevnkrntfgklakrdng odomtimimusnlknyiihvfkdelsisdfekrvaifasyfpiletvdkksikernrtidltlsgrirqfr CCUG 3837emlislvtavdqlrnfythyhhseivienkvldflnsslvstalhvkdkylktdktkefl(NZ_111815535 1)ketiaaeldilieaykkkqiekkntrfkankredilnaiyneafwsfindkdkdketvva >WP_006265509.1kgadayfeknhhksndpdfalnisekgivyllsffltnkemdslkanitgfkgkvdresgnsikymatqrlysfhtyrglkqkirtseegvketllmqmidelskvpnvvyqhlsttqqnsfiedwneyykdyeddvetddlsrvihpvirkryedrfnyfairfldeffdfptlrfqvhlgdyvhdrrtkqlgkvesdriikekvtvfarlkdinsakasyfhsleeqdkeeldnkwtlfpnpsydfpkehtlqhqgeqknagkigiyvklrdtqykekaaleearkslnpkersatkaskydiitqiieandnvksekplvftgqpiaylsmndihsmlfslltdnaelkktpeeveaklidgigkqineilskdtdtkilkkykdndlketdtdkitrdlardkeeieklileqkqraddynytsstkfnidksrkrkhllfnaekgkigvwlandikrfmfkeskskwkgyqhtelqklfayfdtsksdlelilsdmvmvkdypielidlvrksrtivdflnkylearlgyienvitrvknsigtpqfktvrkecfaflkesnytvasldkqierilsmplfiergfmdskptmlegksyqqhkedfadwfvhykensnyqnfydtevyeiitedkreqakvtkkikqqqkndvftlmmvnymleevlklpsndrlslnelyqtkeerivnkqvakdtgernknyiwnkvvdlqlceglvridkvklkdignfrkyendsrvkefltyqsdivwsgylsnevdsnklyvierqldnyesirskellkevqeiecivynqvankeslkqsgnenfkqyvlqgllprgtdvremlilstdvkfkkeeimqlgqvreveqdlysliyirnkfahnqlpikeffdfcennyrpisdneyyaeyymeifrsikekyas (SEQ ID NO: 573) Myroidesmkdilttdttekqnrfyshkiadkyffggyfnlasnniyevfeevnkrntfgklakrdng odomtimimusnlknyiihvfkdelsisdfekrvaifasyfpiletvdkksikernrtidltlsgrirqfr CCUG 12901emlislvtavdqlrnfythyhhseivienkvldflnsslvstalhvkdkylktdktkefl(NZ_H-1590834.1)ketiaaeldilieaykkkqiekkntrfkankredilnaiyneafwsfindkdkdketvva >WP_006261414.1kgadayfeknhhksndpdfalnisekgivyllsffltnkemdslkanitgfkgkvdresgnsikymatqrlysfhtyrglkqkirtseegvketllmqmidelskvpnvvyqhlsttqqnsfiedwneyykdyeddvetddlsrvihpvirkryedrfnyfairfldeffdfptlrfqvhlgdyvhdrrtkqlgkvesdriikekvtvfarlkdinsakanyfhsleeqdkeeldnkwtlfpnpsydfpkehtlqhqgeqknagkigiyvklrdtqykekaaleearkslnpkersatkaskydiitqiieandnvksekplvftgqpiaylsmndihsmlfslltdnaelkktpeeveaklidgigkqineilskdtdtkilkkykdndlketdtdkitrdlardkeeieklileqkqraddynytsstkfnidksrkrkhllfnaekgkigvwlandikrfmteefkskwkgyqhtelqklfayydtsksdldlilsdmvmvkdypielialvkksrtivdflnkylearlgymenvitrvknsigtpqfktvrkecftflkksnytvvsldkqverilsmplfiergfmddkptmlegksyqqhkekfadwfvhykensnyqnfydtevyeittedkrekakvtkkikqqqkndvftlmmvnymleevlklssndrlslnelyqtkeerivnkqvakdtgernknyiwnkvvdlqlceglvridkvklkdignfrkyendsrvkefltyqsdivwsaylsnevdsnklyvierqldnyesirskellkevqeiecsvynqvankeslkqsgnenfkqyvlqglvpigmdvremlilstdvkfikeeiiqlgqageveqdlysliyirnkfahnqlpikeffdfcennyrsisdneyyaeyymeifrsikekyts (SEQ ID NO: 574) Myroidesmkdilttdttekqnrfyshkiadkyffggyfnlasnniyevfeevnkrntfgklakrdng odomtimimusnlknyiihvfkdelsisdfekrvaifasyfpiletvdkksikernrtidltlsgrirqfr CCUG 12901emlislvtavdqlrnfythyhhseivienkvldflnsslvstalhvkdkylktdktkefl(AGED01000033.1)ketiaaeldilieaykkkqiekkntrfkankredilnaiyneafwsfindkdkdketvva >EHO08761.1kgadayfeknhhksndpdfalnisekgivyllsffltnkemdslkanitgfkgkvdresgnsikymatqrlysfhtyrglkqkirtseegvketllmqmidelskvpnvvyqhlsttqqnsfiedwneyykdyeddvetddlsrvihpvirkryedrfnyfairfldeffdfptlrfqvhlgdyvhdrrtkqlgkvesdriikekvtvfarlkdinsakanyfhsleeqdkeeldnkwtlfpnpsydfpkehtlqhqgeqknagkigiyvklrdtqykekaaleearkslnpkersatkaskydiitqiieandnvksekplvftgqpiaylsmndihsmlfslltdnaelkktpeeveaklidgigkqineilskdtdtkilkkykdndlketdtdkitrdlardkeeieklileqkqraddynytsstkfnidksrkrkhllfnaekgkigvwlandikrfmteefkskwkgyqhtelqklfayydtsksdldlilsdmvmvkdypielialvkksrtivdflnkylearlgymenvitrvknsigtpqfktvrkecftflkksnytvvsldkqverilsmplfiergfmddkptmlegksyqqhkekfadwfvhykensnyqnfydtevyeittedkrekakvtkkikqqqkndvftlmmvnymleevlklssndrlslnelyqtkeerivnkqvakdtgernknyiwnkvvdlqlceglvridkvklkdignfrkyendsrvkefltyqsdivwsaylsnevdsnklyvierqldnyesirskellkevqeiecsvynqvankeslkqsgnenfkqyvlqglvpigmdvremlilstdvkfikeeiiqlgqageveqdlysliyirnkfahnqlpikeffdfcennyrsisdneyyaeyymeifrsikekyts (SEQ ID NO: 575) Myroidesmkdilttdttekqnrfyshkiadkyffggyfnlasnniyevfeevnkrntfgklakrdng odomtimimusnlknyiihvfkdelsisdfekrvaifasyfpiletvdkksikernrtidltlsgrirqfr(NZ_CP013690.1)emlislvtavdqlrnfythyhhsdivienkvldflnssfvstalhvkdkylktdktkefl >WP_058700060.1ketiaaeldilieaykkkqiekkntrfkankredilnaiyneafwsfindkdkdkdketvvakgadayfeknhhksndpdfalnisekgivyllsffltnkemdslkanitgfkgkvdresgnsikymatqrlysfhtyrglkqkirtseegvketllmqmidelskvpnvvyqhlsttqqnsfiedwneyykdyeddvetddlsrvthpvirkryedrfnyfairfldeffdfptlrfqvhlgdyvhdrrtkqlgkvesdriikekvtvfarlkdinsakasyfhsleeqdkeeldnkwtlfpnpsydfpkehtlqhqgeqknagkigiyvklrdtqykekaaleearkslnpkersatkaskydiitqiieandnvksekplvftgqpiaylsmndihsmlfslltdnaelkktpeeveaklidgigkqineilskdtdtkilkkykdndlketdtdkitrdlardkeeieklileqkqraddynytsstkfnidksrkrkhllfnaekgkigvwlandikrfmfkeskskwkgyqhielqklfayfdtsksdlelilsnmvmvkdypielidlvkksrtivdflnkylearleyienvitrvknsigtpqfktvrkecftflkksnytvvsldkqverilsmplfiergfmddkptmlegksykqhkekfadwfvhykensnyqnfydtevyeittedkrekakvtkkikqqqkndvftlmmvnymleevlklssndrlslnelyqtkeerivnkqvakdtgernknyiwnkvvdlqlcdglvhidnvklkdignfrkyendsrvkefltyqsdivwsaylsnevdsnklyvierqldnyesirskellkevqeiecsvynqvankeslkqsgnenfkqyvlqgllpigmdvremlilstdvkfkkeeiiqlgqageveqdlysliyirnkfahnqlpikeffdfcennyrsisdneyyaeyymeifrsikekyan (SEQ ID NO: 576) Bergeyellamenktslgnniyynpfkpqdksyfagyfnaamentdsvfrelgkrlkgkeytsenffdai zoohelcumfkenislveyeryvkllsdyfpmarlldkkevpikerkenfkknfkgiikavrdlrnfyt ATCC 43767hkehgeveitdeifgvldemlkstvltvkkkkvktdktkeilkksiekqldilcqkkley(AGYA01000037.1)lrdtarkieekrrnqrergekelvapfkysdkrddliaaiyndafdvyidkkkdslkess >EKB54193.1kakyntksdpqqeegdlkipiskngvvfllslfltkqeihafkskiagfkatvideatvseatvshgknsicfmatheifshlaykklkrkvrtaeinygeaenaeqlsvyaketlmmqmldelskvpdvvyqnlsedvqktfiedwneylkenngdvgtmeeeqvihpvirkryedkfnyfairfldefaqfptlrfqvhlgnylhdsrpkenlisdrrikekitvfgrlselehkkalfikntetnedrehyweifpnpnydfpkenisvndkdfpiagsildrekqpvagkigikvkllnqqyvsevdkavkahqlkqrkaskpsigniieeivpinesnpkeaivfggqptaylsmndihsilyeffdkwekkkeklekkgekelrkeigkelekkivgkiqaqiqqiidkdtnakilkpyqdgnstaidkeklikdlkqeqnilqklkdeqtvrekeyndfiayqdknreinkvrdrnhkqylkdnlkrkypeaparkevlyyrekgkvavwlandikrfmptdfknewkgeqhsllqkslayyeqckeelknllpekvfqhlpfklggyfqqkylyqfytcyldkrleyisglvqqaenfksenkvfkkvenecfkflkkqnythkeldarvqsilgypiflergfmdekptiikgktfkgnealfadwfryykeyqnfqtfydtenyplvelekkqadrkrktkiyqqkkndvftllmakhifksvfkqdsidqfsledlyqsreerlgngerarqtgerntnyiwnktvdlklcdgkitvenvklknvgdfikyeydqrvqaflkyeeniewqaflikeskeeenypyvvereiegyekvrreellkevhlieeyilekvkdkeilkkgdnqnfkyyilngllkqlknedvesykvfnlntepedvninqlkqeatdlegkafvltyirnkfahnqlpkkefwdycqekygkiekektyaeyfaevfkkekealik (SEQ ID NO: 577) Capnocytophagamenktslgnniyynpfkpqdksyfagylnaamenidsvfrelgkrlkgkeytsenffdai cynodegmifkenislveyeryvkllsdyfpmarlldkkevpikerkenfkknfrgiikavrdlrnfyt(NZ_CDOD01000002.1)hkehgeveitdeifgvldemlkstvltvkkkkiktdktkeilkksiekqldilcqkkley >WP_041989581.1lkdtarkieekrrnqrergekklvprfeysdrrddliaaiyndafdvyidkkkdslkessktkyntesypqqeegdlkipiskngvvfllslflskqevhafkskiagfkatvideatvshrknsicfmatheifshlaykklkrkvrtaeinyseaenaeqlsiyaketlmmqmldelskvpdvvyqnlsedvqktfiedwneylkenngdvgtmeeeqvihpvirkryedkfnyfairfldefaqfptlrfqvhlgnylhdsrpkehlisdrrikekitvfgrlselehkkalfikntetnedrkhywevfpnpnydfpkenisvndkdfpiagsildrekqptagkigikvnllnqkyisevdkavkahqlkqrnnkpsigniieeivpingsnpkeiivfggqptaylsmndihsilyeffdkwekkkeklekkgekelrkeigkeleekivgkiqtqiqqiidkdinakilkpyqdddstaidkeklikdlkqeqkilqklkneqtarekeyqeciayqeesrkikrsdksrqkylrnqlkrkypevptrkeilyyqekgkvavwlandikrfmptdfknewkgeqhsllqkslayyeqckeelknllpqqkvfkhlpfelgghfqqkylyqfytryldkrlehisglvqqaenfknenkvfkkvenecfkflkkqnythkgldaqaqsvlgypiflergfmdekptiikgktfkgneslftdwfryykeyqnfqtfydtenyplvelekkqadrkretkiyqqkkndvftllmakhifksvfkqdsidrfsledlyqsreerlengekakqtgerntnyiwnktvdlnlcdgkvtvenvklknvgnfikyeydqrvqtflkyeenikwqaflikeskeeenypyivereieqyekvrreellkevhlieeyilekvkdkeilkkgdnqnfkyyilngllkqlknedvesykvfnlntkpedvninqlkqeatdleqkafvltyirnkfahnqlpkkefwdycqekygkiekektyaeyfaevfkrekealmk (SEQ ID NO: 578) Bergeyellamenktslgnniyynpfkpqdksyfagyfnaamentdsvfrelgkrlkgkeytsenffdai zoohelcumfkenislveyeryvkllsdyfpmarlldkkevpikerkenfkknfkgiikavrdlrnfyt ATCC 43767hkehgeveitdeifgvldemlkstvltvkkkkvktdktkeilkksiekqldilcqkkley(NZ_JH932293.1)lrdtarkieekrrnqrergekelvapfkysdkrddliaaiyndafdvyidkkkdslkess >WP_002664492.1kakyntksdpqqeegdlkipiskngvvfllslfltkqeihafkskiagfkatvideatvseatvshgknsicfmatheifshlaykklkrkvrtaeinygeaenaeqlsvyaketlmmqmldelskvpdvvyqnlsedvqktfiedwneylkenngdvgtmeeeqvihpvirkryedkfnyfairfldefaqfptlrfqvhlgnylhdsrpkenlisdrrikekitvfgrlselehkkalfikntetnedrehyweifpnpnydfpkenisvndkdfpiagsildrekqpvagkigikvkllnqqyvsevdkavkahqlkqrkaskpsigniieeivpinesnpkeaivfggqptaylsmndihsilyeffdkwekkkeklekkgekelrkeigkelekkivgkiqaqiqqiidkdtnakilkpyqdgnstaidkeklikdlkqeqnilqklkdeqtvrekeyndfiayqdknreinkvrdrnhkqylkdnlkrkypeaparkevlyyrekgkvavwlandikrfmptdfknewkgeqhsllqkslayyeqckeelknllpekvfqhlpfklggyfqqkylyqfytcyldkrleyisglvqqaenfksenkvfkkvenecfkflkkqnythkeldarvqsilgypiflergfmdekptiikgktfkgnealfadwfryykeyqnfqtfydtenyplvelekkqadrkrktkiyqqkkndvftllmakhifksvfkqdsidqfsledlyqsreerlgngerarqtgerntnyiwnktvdlklcdgkitvenvklknvgdfikyeydqrvqaflkyeeniewqaflikeskeeenypyvvereiegyekvrreellkevhlieeyilekvkdkeilkkgdnqnfkyyilngllkqlknedvesykvfnlntepedvninqlkqeatdlegkafvltyirnkfahnqlpkkefwdycqekygkiekektyaeyfaevfkkekealik (SEQ ID NO: 579) Flavobacteriummdnnitvektelglgitynhdkvedkhyfggffnlaqnnidlvagefkkrlliqgkdsin sp. 316ifanyfsdqcsitnlergikilaeyfpvvsyidldeknksksirehlillletinnlrny(NZ_JYGZ01000003.1)ythyyhkkiiidgslfplldtillkvvleikkkklkedktkqllkkglekemtilfnlmk >NV_P045968377.1aegkekkikgwnidenikgavinrafshllyndelsdyrkskyntedetlkdtltesgilfllsfflnkkeqeqlkanikgykgkiasipdeeitlknnslrnmathwtyshltykglkhriktdheketllvnmvdylskvpheiyqnlseqnkslfledineymrdneenhdsseasrvihpvirkryenkfayfairfldefaefptlrfmvnvgnyihdnrkkdiggtslitnrtikqqinvfgniteihkkkndyfekeenkektlewelfpnpsyhfqkenipifidleksketndlakeyakekkkifgssrkkqqntakknretiinlvfdkyktsdrktvtfeqptallsfnelnsflyaflvenktgkelekiiiekianqyqilkncsstvdktndnipksikkivntttdsfyfegkkidieklekditieiektnekletikeneesagnykrnerntqkrklyrkyvfftneigieatwitndilrfldnkenwkgyqhselqkfisqydnykkealgllesewnlesdaffgqnlkrmfqsnstfetfykkyldnrkntletylsaienlktmtdvrpkvlkkkwtelfrffdkkiyllstietkinelitkpinlsrgifeekptfingknpnkennqhlfanwfiyakkqtilqdfynlpleqpkaitnlkkhkyklersinnlkiediyikqmvdflyqklfeqsfigslqdlytskekreiekgkakneqtpdesfiwkkgveinthngriiaktkikdigkfknlltdnkiahlisyddriwdfslnndgditkklysintelesyetirrekllkqiqqfeqfllegeteysaerkhpekfekdcnpnfkkyiiegvinkiipnheieeieilkskedvfkinfsdililnndnikkgyllimirnkfahnglidknlfnfslqlysknenenfseylnkvcqniiqefkeklk (SEQ ID NO: 580) Psychroflexusmesiiglglsfnpyktadkhyfgsflnlvennlnavfaefkerisykakdenissliekh torquisfidnmsivdyekkisilngylpiidflddelennlntrvknfkknfiilaeaieklrdyy ATCC 700755thfyhdpitfednkepllelldevllktildvkkkylktdktkeilkdslreemdllvir(NC_018721.1)ktdelrekkktnpkightdssqiknsifndafqgllyedkgnnkktqvshraktrinpkd >WP_015024765.1ihkqeerdfeiplstsglvflmslflskkeiedfksnikgfkgkvvkdenhnslkymathrvysilafkglkyriktdtfsketlmmqmidelskvpdcvyqnlsetkqkdfiedwneyfkdneentenlensrvvhpvirkryedkfnyfairfldefanfktlkfqvfmgyyihdqrtktigttnittertvkekinvfgklskmdnlkkhffsqlsddentdweffpnpsynfltqadnspannipiylelknqqiikekdaikaevnqtqnrnpnkpskrdllnkilktyedfhqgdptailslneipallhlflvkpnnktgqqieniirikiekqfkainhpsknnkgipkslfadtnvrvnaiklkkdleaeldmlnkkhiafkenqkassnydkllkehqftpknkrpelrkyvfyksekgeeatwlandikrfmpkdfktkwkgcqhselqrklafydrhtkqdikellsgcefdhslldinayfqkdnfedffskylenrietlegvlkklhdfkneptplkgvfkncfkflkrqnyvtespeiikkrilakptflprgvfderptmkkgknplkdknefaewfveylenkdyqkfynaeeyrmrdadfkknavikkqklkdfytlqmvnyllkevfgkdemnlqlselfqtrgerlklqgiakkqmnketgdssentrnqtyiwnkdvpvsffngkvtidkvklknigkykryerdervktfigyevdekwmmylphnwkdrysvkpinvidlqiqeyeeirshellkeiqnlegyiydhttdknillqdgnpnfkmyvinglligikqvnipdfivlkqntnfdkidftgiascselekktiiliairnkfahnqlpnkmiydlaneflkieknetyanyylkvlkkmisdla (SEQ ID NO: 581) Flavobacteriummssknesynkqktfnhykqedkyffggflnnaddnlrqvgkefktrinfnhnnnelasvf columnarekdyfnkeksvakrehalnllsnyfpvleriqkhtnhnfeqtreifellldtikklrdyyt ATCC 49512hhyhkpitinpkiydflddtlldvlitikkkkvkndtsrellkeklrpeltqlknqkree(NC_016510.2)likkgkklleenlenavfnhclrpfleenktddkqnktvslrkyrkskpneetsitltqs >WP_014165541.1glvflmsfflhrkefqvftsglegfkakvntikeeeislnknnivymithwsysyynfkglkhriktdqgvstleqnntthsltntntkealltqivdylskvpneiyetlsekqqkefeedineymrenpenedstfssivshkvirkryenkfnyfamrfldeyaelptlrfmvnfgdyikdrqkkilesiqfdseriikkeihlfeklslvteykknvylketsnidlsrfplfpnpsyvmannnipfyidsrsnnldeylnqkkkaqsqnkkrnitfekynkeqskdaiiamlqkeigvkdlqqrstigllscnelpsmlyevivkdikgaelenkiaqkireqyqsirdftldspqkdnipttliktintdssvtfenqpidiprlknaigkeltltqekllnvkeheievdnynrnkntykfknqpknkvddkklqrkyvfyrneirgeanwlasdlihfmknkslwkgymhnelqsflaffedkkndcialletvfnlkedciltkglknlflkhgnfidfykeylklkedflntestflengliglppkilkkelskrfkyifivfqkrqfiikeleekknnlyadainlsrgifdekptmipfkkpnpdefaswfvasyqynnyqsfyeltpdiverdkkkkyknlrainkvkiqdyylklmvdtlyqdlfnqpldkslsdfyvskaerekikadakayqkrndsslwnkvihlslqnnritanpklkdigkykralqdekiatlltyddrtwtyalqkpekenendykelhytalnmelqeyekvrskellkqvqelekqileeytdflstqihpadferegnpnfkkylahsileneddldklpekveamreldetitnpiikkaivliiirnkmahnqyppkfiydlanrfvpkkeeeyfatyfnrvfetitkelwenkekkdktqv (SEQ ID NO: 582) Flavobacteriummssknesynkqktfnhykqedkyffggflnnaddnlrqvgkefktrinfnhnnnelasvf columnarekdyfnkeksvakrehalnllsnyfpvleriqkhtnhnfeqtreifellldtikklrdyyt(NZ_CP013992.1)hhyhkpitinpkvydflddtlldvlitikkkkvkndtsrellkekfrpeltqlknqkree >WP_060381855.1likkgkklleenlenavfnhclrpfleenktddkqnktvslrkyrkskpneetsitltqsglvflisfflhrkefqvftsglegfkakvntikeeeislnknnivymithwsysyynfkglkhriktdqgvstleqnntthsltntntkealltqivdylskvpneiyetlsekqqkefeedineymrenpenedstfssivshkvirkryenkfnyfamrfldeyaelptlrfmvnfgdyikdrqkkilesiqfdseriikkeihlfeklglvteykknvylketsnidlsrfplfpspsyvmannnipfyidsrsnnldeylnqkkkaqsqnrkrnitfekynkeqskdaiiamlqkeigvkdlqqrstigllscnelpsmlyevivkdikgaelenkiaqkireqyqsirdftldspqkdnipttltktistdtsvtfenqpidiprlknalqkeltltqekllnvkqheievdnynrnkntykfknqpkdkvddnklqrkyvfyrneiggeanwlasdlihfmknkslwkgymhnelqsflaffedkkndcialletvfnlkedciltkdlknlflkhgnfidfykeylklkedflntestflengfiglppkilkkelskrinyifivfqkrqfiikeleekknnlyadainlsrgifdekptmipfkkpnpdefaswfvasyqynnyqsfyeltpdkiendkkkkyknlrainkvkiqdyylklmvdtlyqdlfnqpldkslsdfyvsktdrekikadakayqkrndsflwnkvihlslqnnritanpklkdigkykralqdekiatlltyddrtwtyalqkpekenendykelhytalnmelqeyekvrskkllkqvqelekqildkfydfsnnathpedleiedkkgkrhpnfklyitkallkneseiinlenidieilikyydynteklkekiknmdedekakivntkenynkitnvlikkalvliiirnkmahnqyppkfiydlatrfvpkkeeeyfacyfnrvfetittelwenkkkakeiv (SEQ ID NO: 583) Flavobacteriummssknesynkqktfnhykqedkyffggflnnaddnlrqvgkefktrinfnhnnnelasvf columnarekdyfnkeksvakrehalnllsnyfpvleriqkhtnhnfeqtreifellldtikklrdyyt(NZ_CP015107.1)hhyhkpitinpkiydflddtlldvlitikkkkvkndtsrellkeklrpeltqlknqkree >WP_063744070.1likkgkklleenlenavfnhclrpfleenktddkqnktvslrkyrkskpneetsitltqsglvflmsfflhrkefqvftsglegfkakvntikeekislnknnivymithwsysyynfkglkhriktdqgvstleqnntthsltntntkealltqivdylskvpneiyetlsekqqkefeedineymrenpenedstfssivshkvirkryenkfnyfamrfldeyaelptlrfmvnfgdyikdrqkkilesiqfdseriikkeihlfeklglvteykknvylketsnidlsrfplfpspsyvmannnipfyidsrsnnldeylnqkkkaqsqnrkrnitfekynkeqskdaiiamlqkeigvkdlqqrstigllscnelpsmlyevivkdikgaelenkiaqkireqyqsirdftlnspqkdnipttliktistdtsvtfenqpidiprlknaigkelaltqekllnvkqheievnnynrnkntykfknqpkdkvddnklqrkyvfyrneiggeanwlasdlihfmknkslwkgymhnelqsflaffedkkndcialletvfnlkedciltkdlknlflkhgnfidfykeylklkedflntestflengfiglppkilkkelskrinyifivfqkrqfiikeleekknnlyadainlsrgifdekptmipfkkpnpdefaswfvasyqynnyqsfyeltpdkiendkkkkyknlrainkvkiqdyylklmvdtlyqdlfnqpldkslsdfyvsktdrekikadakayqkrndsflwnkvihlslqnnritanpklkdigkykralqdekiatlltyddrtwtyalqkpekenendykelhytalnmelqeyekvrskkllkqvqelekqildkfydfsnnathpedleiedkkgkrhpnfklyitkallkneseiinlenidieilikyydynteklkekiknmdedekakivntkenynkitnvlikkalvliiirnkmahnqyppkfiydlatrfvpkkeeeyfacyfnrvfetittelwenkkkakeiv (SEQ ID NO: 584) Flavobacteriummssknesynkqktfnhykqedkyffggflnnaddnlrqvgkefktrinfnhnnnelasvf columnarekdyfnkeksvakrehalnllsnyfpvleriqkhtnhnfeqtreifellldtikklrdyyt(NZ_CP016277.1)hhyhkpitinpkiydflddtlldvlitikkkkvkndtsrellkeklrpeltqlknqkree >WP_065213424.1likkgkklleenlenavfnhclipfleenktddkqnktvslrkyrkskpneetsitltqsglvflmsfflhrkefqvftsglerfkakvntikeeeislnknnivymithwsysyynfkglkhriktdqgvstleqnntthsltntntkealltqivdylskvpneiyetlsekqqkefeedineymrenpenedstfssivshkvirkryenkfnyfamrfldeyaelptlrfmvnfgdyikdrqkkilesiqfdseriikkeihlfeklslvteykknvylketsnidlsrfplfpnpsyvmannnipfyidsrsnnldeylnqkkkaqsqnkkrnitfekynkeqskdaiiamlqkeigvkdlqqrstigllscnelpsmlyevivkdikgaelenkiaqkireqyqsirdftldspqkdnipttliktintdssvtfenqpidiprlknalqkeltltqekllnvkeheievdnynrnkntykfknqpknkvddkklqrkyvfyrneirgeanwlasdlihfmknkslwkgymhnelqsflaffedkkndcialletvfnlkedciltkglknlflkhgnfidfykeylklkedflstestflengfiglppkilkkelskrlkyifivfqkrqfiikeleekknnlyadainlsrgifdekptmipfkkpnpdefaswfvasyqynnyqsfyeltpdiverdkkkkyknlrainkvkiqdyylklmvdtlyqdlfnqpldkslsdfyvskaerekikadakayqklndsslwnkvihlslqnnritanpklkdigkykralqdekiatlltydartwtyalqkpekenendykelhytalnmelqeyekvrskellkqvqelekkildkfydfsnnashpedleiedkkgkrhpnfklyitkallkneseiinlenidieillkyydynteelkekiknmdedekakiintkenynkitnvlikkalvliiirnkmahnqyppkfiydlanrfvpkkeeeyfatyfnrvfetitkelwenkekkdktqv (SEQ ID NO: 585) Chryseobacteriummetqtighgiaydhskiqdkhffggflnlaennikavlkafsekfnvgnvdvkqfadvsl sp. YR477kdnlpdndfqkrvsflkmyfpvvdfinipnnrakfrsdlttlfksvdqlrnfythyyhkp(NZ_KN549099.1)ldfdaslfillddifartakevrdqkmkddktrqllskslseelqkgyelqlerlkelnr >WP_047431796.1lgkkvnihdqlgikngvinnafnhliykdgesfktkltyssaltsfesaengieisqsgllfllsmflkrkeiedlknrnkgfkakvvidedgkvnglkfmathwvfsylcfkglksklstefheetlliqiidelskvpdelycafdketrdkfiedineyvkeghqdfsledakvihpvirkryenkfnyfairfldefvkfpslrfqvhvgnyvhdrriknidgttfetervvkdrikvfgrlseissykagylssysdkhdetgweifpnpsyvfinnnipihisvdtsfkkeiadfkklrraqvpdelkirgaekkrkfeitqmigsksvinqeepiallslneipallyeilingkepaeieriikdklnerqdviknynpenwlpasqisrrlrsnkgeriintdkllqlvtkellvteqklkiisdnrealkqkkegkyirkfiftnselgreaiwladdikrfmpadvrkewkgyqhsqlqqslafynsrpkealailesswnlkdekiiwnewilksftqnkffdafyneylkgrkkyfaflsehivqytsnaknlqkfikqqmpkdlfekrhyliedlqteknkilskpfifprgifdkkptfikgvkvedspesfanwyqygyqkdhqfqkfydwkrdysdvflehlgkpfinngdrrtlgmeelkeriiikqdlkikkikiqdlflrliaenlfqkvfkysaklplsdfyltqeermekenmaalqnvreegdkspniikdnfiwskmipykkgqiienavklkdigklnvlslddkvqtllsyddakpwskialenefsigensyevirreklfkeiqqfeseilfrsgwdginhpaqlednrnpkfkmyivngilrksaglysqgediwfeynadfnnldadvletkselvqlaflvtairnkfahnqlpakefyfyirakygfadepsvalvylnftkyainefkkvmi (SEQ ID NO: 586) RiemerellamekpllpnvytlkhkffwgaflniarhnafitichineqlglktpsnddkivdvvcetwnanatipestifernilnndhdllkksqltelilkhfpfltamcyhppkkegkkkghqkeqqkekeseaqsqaeATCC 11845 = DSMalnpskliealeilvnqlhslrnyyshykhkkpdaekdifkhlykafdaslrmvkedyka 15868 hftvnitrdfahlnrkgknkqdnpdfnryrfekdgfftesgllfftnlfldkrdaywmlk(NC_014738.1)kvsgfkashkgrekmttevfcrsrillpklrlesrydhnqmlldmlselsrcpkllyekl >WP_004919755Aseenkkhfqveadgfldeieeeqnpfkdtlirhqdrfpyfalryldlnesfksirfqvdlgtyhyciydkkigdeqekrhltrtllsfgrlqdfteinrpqewkaltkdldyketsnqpfiskttphyhitdnkigfrlgtskelypsleikdganriakypynsgfvahafisvhellplmfyqhltgksedllketvrhiqriykdfeeerintiedlekanqgrlplgafpkqmlgllqnkqpdlsekakikiekliaetkllshrintklksspklgkrrekliktgvladwlvkdfmrfqpvaydaqnqpiksskanstefwfirralalyggeknrlegyfkqtnligntnphpflnkfnwkacrnlvdfyqqylegrekfleaiknqpwepyqyclllkipkenrknlvkgweqggislprglfteairetlsedlmlskpirkeikkhgrvgfisraitlyfkekyqdkhqsfynlsykleakapllkreehyeywqqnkpqsptesqrlelhtsdrwkdyllykrwqhlekklrlyrnqdvmlwlmtleltknhfkelnlnyhqlklenlavnvqeadaklnpinqtlpmvlpvkvypatafgevqyhktpirtvyireehtkalkmgnfkalvkdrringlfsfikeendtqkhpisqlrlrreleiyqslrvdafketlsleekllnkhtslsslenefralleewkkeyaassmvtdehiafiasvrnafchnqypfykealhapiplftvaqptteekdglgiaeallkvlreyceivksqi (SEQ ID NO: 587) Riemerellamffsfhnaqrvifkhlykafdaslrmvkedykahftvnitrdfahlnrkgknkqdnpdfnanatipestiferryrfekdgfftesgllfftnlfldkrdaywmlkkvsgfkashkgrekmttevfcrsrill RA-CH-2pklrlesrydhnqmlldmlselsrcpkllyeklseenkkhfqveadgfldeieeeqnpfk(NC_020125.1)dtlirhqdrfpyfalryldlnesfksirfqvdlgtyhyciydkkigdeqekrhltrtlls >WP_015345620Afgrlqdfteinrpqewkaltkdldyketsnqpfiskttphyhitdnkigfrlgtskelypsleikdganriakypynsgfvahafisvhellplmfyqhltgksedllketvrhiqriykdfeeerintiedlekanqgrlplgafpkwalgllqnkqpdlsekakikiekliaetkllshrintklksspklgkrrekliktgvladwlvkdfmrfqpvaydaqnqpiksskanstefwfirralalyggeknrlegyfkqtnligntnphpflnkfnwkacrnlvdfyqqylegrekfleaikhqpwepyqyclllkvpkenrknlvkgweqggislprglfteairetlskdltlskpirkeikkhgrvgfisraitlyfkekyqdkhqsfynlsykleakapllkkeehyeywqqnkpqsptesqrlelhtsdrwkdyllykrwqhlekklrlyrnqdimlwlmtleltknhfkelnlnyhqlklenlavnvqeadaklnpinqtlpmvlpvkvypttafgevqyhetpirtvyireeqtkalkmgnfkalvkdrringlfsfikeendtqkhpisqlrlrreleiyqslrvdafketlsleekllnkhaslsslenefrtlleewkkkyaassmvtdkhiafiasvrnafchnqypfyketlhapillftvaqptteekdglgiaeallkvlreyceivksqi (SEQ ID NO: 588)Riemerella mffsfhnaqrvifkhlykafdaslrmvkedykahftvnitrdfahlnrkgknkqdnpdfnanatipestiferryrfekdgfftesgllfftnlfldkrdaywmlkkvsgfkashkgrekmttevfcrsrill(NZ_CP007504.1)pklrlesrydhnqmlldmlselsrcpkllyeklseenkkhfqveadgfldeieeeqnpfk >WP_049354263.1dtlirhqdrfpyfalryldlnesfksirfqvdlgtyhyciydkkigdeqekrhltrtllsfgrlqdfteinrpqewkaltkdldyketsnqpfiskttphyhitdnkigfrlgtskelypsleikdganriakypynsgfvahafisvhellplmfyqhltgksedllketvrhiqriykdfeeerintiedlekanqgrlplgafpkwalgllqnkqpdlsekakikiekliaetkllshrintklksspklgkrrekliktgvladwlvkdfmrfqpvaydaqnqpiksskanstefwfirralalyggeknrlegyfkqtnligntnphpflnkfnwkacrnlvdfyqqylegrekfleaiknqpwepyqyclllkipkenrknlvkgweqggislprglfteairetlsedlmlskpirkeikkhgrvgfisraitlyfkekyqdkhqsfynlsykleakapllkreehyeywqqnkpqsptesqrlelhtsdrwkdyllykrwqhlekklrlyrnqdvmlwlmtleltknhfkelnlnyhqlklenlavnvqeadaklnpinqtlpmvlpvkvypatafgevqyhktpirtvyireehtkalkmgnfkalvkdrringlfsfikeendtqkhpisqlrlrreleiyqslrvdafketlsleekllnkhtslsslenefralleewkkeyaassmvtdehiafiasvrnafchnqypfykealhapiplftvaqptteekdglgiaeallkvlreyceivksqi (SEQ ID NO:  589)Riemerella mffsfhnaqrvifkhlykafdaslrmvkedykahftvnitrdfahlnrkgknkqdnpdfnanatipestiferryrfekdgfftesgllfftnlfldkrdaywmlkkvsgfkashkqsekmttevfcrsrill(NZ_LUDU01000012.1)pklrlesrydhnqmlldmlselsrcpkllyeklsekdkkcfqveadgfldeieeeqnpfk >WP_061710138Adtlirhqdrfpyfalryldlnesfksirfqvdlgtyhyciydkkigyegekrhltrtllnfgrlqdfteinrpqewkaltkdldynetsnqpfiskttphyhitdnkigfrlrtskelypslevkdganriakypynsdfvahafisisvhellplmfyqhltgksedllketvrhiqriykdfeeerintiedlekanqgrlplgafpkwalgllqnkqpdlsekakikiekliaetkllshrintklksspklgkrrekliktgvladwlvkdfmrfqpvvydaqnqpiksskanstesrlirralalyggeknrlegyfkqtnligntnphpflnkfnwkacrnlvdfyqqylegrekfleaikhqpwepyqyclllkvpkenrknlvkgweqggislprglfteairetlskdltlskpirkeikkhgrvgfisraitlyfkekyqdkhqsfynlsykleakapllkkeehyeywqqnkpqsptesqrlelhtsdrwkdyllykrwqhlekklrlyrnqdimlwlmtleltknhfkelnlnyhqlklenlavnvqeadaklnpinqtlpmvlpvkvypttafgevqyhetpirtvyireeqtkalkmgnfkalvkdrhinglfsfikeendtqkhpisqlrlrreleiyqslrvdafketlsleekllnkhaslsslenefrtlleewkkkyaassmvtdkhiafiasvrnafchnqypfyketlhapillftvaqptteekdglgiaeallrvlreyceivksqi (SEQ ID NO: 590)Riemerella mekplppnvytlkhkffwgaflniarhnafitichineqlglttppnddkiadvvcgtwnanatipestifernilnndhdllkksqltelilkhfpflaamcyhppkkegkkkgsqkeqqkekeneaqsqae(NZ_LUDI01000010.1)alnpselikvlktivkqlrtlrnyyshhshkkpdaekdifkhlykafdaslrmvkedyka >WP_064970887.1hftvnitqdfahlnrkgknkqdnpdfdryrfekdgfftesgllfftnlfldkrdaywmlkkvsgfkashkgsekmttevfcrsrillpklrlesrydhnqmlldmlselsrypkllyeklseedkkrfqveadgfldeieeeqnpfkdtlirhqdrfpyfalryldlnesfksirfqvdlgtyhyciydkkigdeqekrhltrtllsfgrlqdfteinrpqewkaltkdldyketskqpfiskttphyhitdnkigfrlgtskelypslevkdganriaqypynsdfvahafisvhellplmfyqhltgksedllketvrhiqriykdfeeerintiedlekanqgrlplgafpkqmlgllqnkqpdlsekakikiekliaetkllshrintklksspklgkrrekliktgvladwlvkdfmrfqpvaydaqnqpiesskanstefqliqralalyggeknrlegyfkqtnligntnphpflnkfnwkacrnlvdfyqqylegrekfleaiknqpwepyqyclllkipkenrknlvkgweqggislprglfteairetlskdltlskpirkeikkhgrvgfisraitlyfrekyqddhqsfydlpykleakasplpkkehyeywqqnkpqsptelqrlelhtsdrwkdyllykrwqhlekklrlyrnqdvmlwlmtleltknhfkelnlnyhqlklenlavnvqeadaklnpinqtlpmvlpvkvypatafgevqygetpirtvyireeqtkalkmgnfkalvkdrringlfsfikeendtqkhpisqlrlrreleiyqslrvdafketlnleekllkkhtslssvenkfrilleewkkeyaassmvtdehiafiasvrnafchnqypfyeealhapiplftvaqqtteekdglgiaeallrvlreyceivksqi (SEQ ID NO: 591) Prevotellammekenvqgshiyyeptdkcfwaafynlarhnayltiahinsfvnskkginnddkvldiisaccharolyticaddwskfdndllmgarinklilkhfpflkaplyglakrktrkqqgkeqqdyekkgdedpev F0055igeaianafkmanvrktlhaflkqledlrnhfshynynspakkmevkfddgfcnklyyvfAMEP01000091.1)daalqmvkddnrmnpeinmqtdfehlvrlgrnrkipntfkynftnsdgtinnngllffvs >EKY00089.1lflekrdaiwmqkkikgfkggtenymrmtnevfcrnrmvipklrletdydnhqlmfdmlnelvrcplslykrlkqedqdkfrvpiefldedneadnpygenansdenpteetdplkntivrhqhrfpyfvlryfdlnevfkqlrfqinlgcyhfsiydktigertekrhltrtlfgfdrlqnfsvklqpehwknmvkhldteessdkpylsdamphyqienekigihflktdtekketvwpsleveevssnrnkykseknitadaflsthellpmmfyyqllsseektraaagdkvqgvlqsyrkkifdiyddfangtinsmqklderlakdnllrgnmpqqmlailehqepdmegkakekldrlitetkkrigkledqfkqkvrigkrradlpkvgsiadwlvndmmrfqpakrnadntgvpdskansteyrllgealafysaykdrlepyfrqvnliggtnphpflhrvdwkkcnhllsfyhdyleakeqylshlspadwqkhqhflllkvrkdignekkdwkkslvagwkngfnlprglftesiktwfstdadkvqitdtklfenrvgliakliplyydkvyndkpqpfyqypfnindrykpedtrkrftaassklwnekkmlyknaqpdssdkieypqyldflswkklerelrmlrnqdmmvwlmckdlfaqctvegvefadlklsqlevdvnvqdnlnvinnvssmilplsvypsdaqgnvlrnskplhtvyvqenntkllkqgnfksllkdrringlfsfiaaegedlqqhpltknrleyelsiyqtmrisvfeqtlqlekailtrnkticgnnfnnllnswsehrtdkktlqpdidfliavrnafshnqypmstntvmqgiekfniqtpkleekdglgiasqlakktkdaasrlqniinggtn (SEQ ID NO: 592) Prevotellamedkpfwaaffnlarhnvyltvnhinklldleklydegkhkeiferedifnisddvmndasaccharolyticansngkkrkldikkiwddldtdltrkyqlrelilkhfpfiqpaiigaqtkerttidkdkrs JCM 17484tstsndslkqtgegdindllslsnvksmffrllqileqlrnyyshvkhsksatmpnfded(NZ_BAKN01000001.1)llnwmryifidsvnkvkedyssnsvidpntsfshliykdeqgkikperypftskdgsina >WP_051522484.1fgllffvslflekqdsiwmqkkipgfkkasenymkmtnevfcrnhillpkirletvydkdwmlldmlnevvrcplslykrltpaaqnkfkvpekssdnanrqeddnpfsrilvrhqnrfpyfvlrffdlnevfttlrfqinlgcyhfaickkqigdkkevhhlirtlygfsrlqnftqntrpeewntivkttepssgndgktvqgvplpyisytiphyqienekigikifdgdtavdtdiwpsystekqlnkpdkytltpgfkadvflsvhellpmmfyyqlllcegmlktdagnavekvlidtrnaifnlydafvqekintitdlenylqdkpilighlpkqmidllkghqrdmlkaveqkkamlikdterrlklldkqlkqetdvaakntgtllkngqiadwlvndmmrfqpvkrdkegnpincskansteyqmlqrafafyatdscrlsryftqlhlihsdnshlflsrfeydkqpnliafyaaylkakleflnelqpqnwasdnyflllrapkndrqklaegwkngfnlprglftekiktwfnehktivdisdcdifknrvgqvarlipvffdkkfkdhsqpfyrydfnvgnvskpteanylskgkreelfksyqnkfknnipaektkeyreyknfslwkkferelrliknqdiliwlmcknlfdekikpkkdilepriaysyikldslqtntstagslnalakvvpmtlaihidspkpkgkagnnekenkeftvyikeegtkllkwgnfktlladrrikglfsyiehddidlkqhpltkrrvdleldlyqtcridifqqtlgleaqlldkysdlntdnfyqmligwrkkegiprnikedtdflkdvrnafshnqypdskkiafrrirkfnpkelileeeeglgiatqmykevekvvnrikrielfd (SEQ ID NO: 593) Prevotella buccaemqkqdklfvdrkknaifafpkyitimenkekpepiyyeltdkhfwaaflnlarhnvytti ATCC 33574nhinrrleiaelkddgymmgikgswneqakkldkkvrlrdlimkhfpfleaaayemtnsk(AEPD01000005.1)spnnkegrekeqsealslnnlknvlfifleklqvlrnyyshykyseespkpifetsllkn >EFU31981.1mykvfdanvrlvkrdymhhenidmqrdfthlnrkkqvgrtkniidspnfhyhfadkegnmtiagllffvslfldkkdaiwmqkklkgfkdgrnlreqmtnevfcrsrislpklklenvqtkdwmqldmlnelvrcpkslyerlrekdresfkvpfdifsddynaeeepfkntivrhqdrfpyfvlryfdlneifeqlrfqidlgtyhfsiynkrigdedevrhlthhlygfariqdfapqnqpeewrklvkdldhfetsgepyisktaphyhlenekigikfcsahnnlfpslqtdktcngrskfnlgtqftaeaflsvhellpmmfyyllltkdysrkesadkvegiirkeisniyaiydafanneinsiadltrrlqntnilqghlpkqmisilkgrqkdmgkeaerkigemiddtqrrldllckqtnqkirigkrnagllksgkiadwlvndmmrfqpvqkdqnnipinnskansteyrmlqralalfgsenfrlkayfnqmnlvgndnphpflaetqwehqtnilsfyrnylearkkylkglkpqnwkqyqhflilkvqktnrntivtgwknsfnlprgiftqpirewfekhnnskriydgilsfdrvgfvakaiplyfaeeykdnvqpfydypfnignrlkpkkrqfldkkervelwqknkelfknypsekkktdlayldflswkkferelrliknqdivtwlmfkelfnmatveglkigeihlrdidtntaneesnnilnrimpmklpvktyetdnkgnilkerplatfyieetetkvlkqgnfkalvkdrringlfsfaettdlnleehpisklsvdlelikyqttrisifemtlglekklidkystlptdsfrnmlerwlqckanrpelknyvnsliavrnafshnqypmydatlfaevkkftlfpsvdtkkielniapqlleivgkaikeieksenkn (SEQ ID NO:  594)Prevotella buccaemqkqdklfvdrkknaifafpkyitimenkekpepiyyeltdkhfwaaflnlarhnvytti ATCC 33574nhinrrleiaelkddgymmgikgswneqakkldkkvrlrdlimkhfpfleaaayemtnsk(NZ_GL586311.1)spnnkegrekeqsealslnnlknvlfifleklqvlrnyyshykyseespkpifetsllkn >WP_004343973.1mykvfdanvrlvkrdymhhenidmqrdfthlnrkkqvgrtkniidspnfhyhfadkegnmtiagllffvslfldkkdaiwmqkklkgfkdgrnlreqmtnevfcrsrislpklklenvqtkdwmqldmlnelvrcpkslyerlrekdresfkvpfdifsddynaeeepfkntivrhqdrfpyfvlryfdlneifeqlrfqidlgtyhfsiynkrigdedevrhlthhlygfariqdfapqnqpeewrklvkdldhfetsgepyisktaphyhlenekigikfcsahnnlfpslqtdktcngrskfnlgtqftaeaflsvhellpmmfyyllltkdysrkesadkvegiirkeisniyaiydafanneinsiadltrrlqntnilqghlpkqmisilkgrqkdmgkeaerkigemiddtqrrldllckqtnqkirigkrnagllksgkiadwlvndmmrfqpvqkdqnnipinnskansteyrmlqralalfgsenfrlkayfnqmnlvgndnphpflaetqwehqtnilsfyrnylearkkylkglkpqnwkqyqhflilkvqktnrntivtgwknsfnlprgiftqpirewfekhnnskriydgilsfdrvgfvakaiplyfaeeykdnvqpfydypfnignrlkpkkrqfldkkervelwqknkelfknypsekkktdlayldflswkkferelrliknqdivtwlmfkelfnmatveglkigeihlrdidtntaneesnnilnrimpmklpvktyetdnkgnilkerplatfyieetetkvlkqgnfkalvkdrringlfsfaettdlnleehpisklsvdlelikyqttrisifemtlglekklidkystlptdsfrnmlerwlqckanrpelknyvnsliavrnafshnqypmydatlfaevkkftlfpsvdtkkielniapqlleivgkaikeieksenkn (SEQ ID NO:  595)Prevotella buccaemqkqdklfvdrkknaifafpkyitimengekpepiyyeltdkhfwaaflnlarhnvytti D17nhinrrleiaelkddgymmdikgswneqakkldkkvrlrdlimkhfpfleaaayeitnsk(NZ_GG739967.1)spnnkegrekeqsealslnnlknvlfifleklqvlrnyyshykyseespkpifetsllkn >WP_004343581.1mykvfdanvrlvkrdymhhenidmqrdfthlnrkkqvgrtkniidspnfhyhfadkegnmtiagllffvslfldkkdaiwmqkklkgfkdgrnlreqmtnevfcrsrislpklklenvqtkdwmqldmlnelvrcpkslyerlrekdresfkvpfdifsddydaeeepfkntivrhqdrfpyfvlryfdlneifeqlrfqidlgtyhfsiynkrigdedevrhlthhlygfariqdfaqqnqpevwrklvkdldyfeasqepyipktaphyhlenekigikfcsthnnlfpslktektcngrskfnlgtqftaeaflsvhellpmmfyyllltkdysrkesadkvegiirkeisniyaiydafangeinsiadltcrlqktnilqghlpkqmisilegrqkdmekeaerkigemiddtqrrldllckqtnqkirigkrnagllksgkiadwlvndmmrfqpvqkdqnnipinnskansteyrmlqralalfgsenfrlkayfnqmnlvgndnphpflaetqwehqtnilsfyrnylearkkylkglkpqnwkqyqhflilkvqktnrntivtgwknsfnlprgiftqpirewfekhnnskriydgilsfdrvgfvakaiplyfaeeykdnvqpfydypfnignklkpqkgqfldkkervelwqknkelfknypsekkktdlayldflswkkferelrliknqdivtwlmfkelfnmatveglkigeihlrdidtntaneesnnilnrimpmklpvktyetdnkgnilkerplatfyieetetkvlkqgnfkvlakdrringllsfaettdidleknpitklsvdhelikyqttrisifemtlglekklinkyptlptdsfrnmlerwlqckanrpelknyvnsliavrnafshnqypmydatlfaevkkftlfpsvdtkkielniapqlleivgkaikeieksenkn (SEQ ID NO:  596)Prevotella mqkqdklfvdrkknaifafpkyitimengekpepiyyeltdkhfwaaflnlarhnvyttisp. MSX73 nhinrrleiaelkddgymmgikgswneqakkldkkvrlrdlimkhfpfleaaayeitnsk(NZ_ALJQ01000043.1)spnnkegrekeqsealslnnlknvlfifleklqvlrnyyshykyseespkpifetsllkn >W1_007412163.1mykvfdanvrlvkrdymhhenidmqrdfthlnrkkqvgrtkniidspnfhyhfadkegnmtiagllffvslfldkkdaiwmqkklkgfkdgrnlreqmtnevfcrsrislpklklenvqtkdwmqldmlnelvrcpkslyerlrekdresfkvpfdifsddydaeeepfkntivrhqdrfpyfvlryfdlneifeqlrfqidlgtyhfsiynkrigdedevrhlthhlygfariqdfapqnqpeewrklvkdldhfetsgepyisktaphyhlenekigikfcsthnnlfpslkrektcngrskfnlgtqftaeaflsvhellpmmfyyllltkdysrkesadkvegiirkeisniyaiydafanneinsiadltcrlqktnilqghlpkqmisilegrqkdmekeaerkigemiddtqrrldllckqtnqkirigkrnagllksgkiadwlvsdmmrfqpvqkdtnnapinnskansteyrmlqhalalfgsessrlkayfrqmnlvgnanphpflaetqwehqtnilsfyrnylearkkylkglkpqnwkqyqhflilkvqktnrntivtgwknsfnlprgiftqpirewfekhnnskriydgilsfdrvgfvakaiplyfaeeykdnvqpfydypfnignklkpqkgqfldkkervelwqknkelfknypseknktdlayldflswkkferelrliknqdivtwlmfkelfktttveglkigeihlrdidtntaneesnnilnrimpmklpvktyetdnkgnilkerplatfyieetetkvlkqgnfkvlakdrringllsfaettdidleknpitklsvdyelikyqttrisifemtlglekklidkystlptdsfrnmlerwlqckanrpelknyvnsliavrnafshnqypmydatlfaevkkftlfpsvdtkkielniapqlleivgkaikeieksenkn (SEQ ID NO:  597)Prevotella pallensmkeeekgktpvvstynkddkhfwaaflnlarhnvyitvnhinkilgegeinrdgyentle ATCC 700821kswneikdinkkdrlskliikhfpflevttyqrnsadttkqkeekqaeaqsleslkksff(AFPY01000052.1)vfiyklrdlrnhyshykhskslerpkfeedlqekmynifdasiqlvkedykhntdiktee >EGQ18444.1dfkhldrkgqfkysfadnegnitesgllffvslflekkdaiwvqkklegfkcsnesyqkmtnevfcrsrmllpklrlqstqtqdwilldmlnelircpkslyerlreedrkkfrvpieiadedydaeqepfknalvrhqdrfpyfalryfdyneiftnlrfqidlgtyhfsiykkqigdykeshhlthklygferigeftkqnrpdewrkfvktfnsfetskepyipettphyhlenqkigirfrndndkiwpslktnseknekskykldksfqaeaflsvhellpmmfyylllktentdndneietkkkenkndkqekhkieeiienkiteiyalydafangkinsidkleeyckgkdieighlpkqmiailksehkdmateakrkqeemladvqkslesldnqineeienverknsslksgeiaswlvndmmrfqpvqkdnegnpinnskansteyqmlqrslalynkeekptryfrqvnliessnphpflnntewekcnnilsfyrsyleakknfleslkpedweknqyflmlkepktncetivqgwkngfnlprgiftepirkwfmehrknitvaelkrvglvakviplffseeykdsvqpfynylfnvgninkpdeknflnceerrellrkkkdefkkmtdkekeenpsylefqswnkferelrlvrnqdivtwllcmelfnkkkikelnvekiylknintnttkkeknteekngeekiikeknnilnrimpmrlpikvygrenfsknkkkkirrntfftvyieekgtkllkqgnfkalerdrrlgglfsfvkthskaesksntisksrveyelgeyqkarieiikdmlaleetlidkynsldtdnfhnmltgwlklkdepdkasfqndvdlliavrnafshnqypmrnriafaninpfslssantseekglgianqlkdkthktiekiieiekpietke (SEQ ID NO:  598)Prevotella pallensmkeeekgktpvvstynkddkhfwaaflnlarhnvyitvnhinkilgegeinrdgyentle ATCC 700821kswneikdinkkdrlskliikhfpflevttyqrnsadttkqkeekqaeaqsleslkksff(NZ_GL982513.1)vfiyklrdlrnhyshykhskslerpkfeedlqekmynifdasiqlvkedykhntdiktee >WP_006044833.1dfkhldrkgqfkysfadnegnitesgllffvslflekkdaiwvqkklegfkcsnesyqkmtnevfcrsrmllpklrlqstqtqdwilldmlnelircpkslyerlreedrkkfrvpieiadedydaeqepfknalvrhqdrfpyfalryfdyneiftnlrfqidlgtyhfsiykkqigdykeshhlthklygferigeftkqnrpdewrkfvktfnsfetskepyipettphyhlenqkigirfrndndkiwpslktnseknekskykldksfqaeaflsvhellpmmfyylllktentdndneietkkkenkndkqekhkieeiienkiteiyalydafangkinsidkleeyckgkdieighlpkqmiailksehkdmateakrkqeemladvqkslesldnqineeienverknsslksgeiaswlvndmmrfqpvqkdnegnpinnskansteyqmlqrslalynkeekptryfrqvnliessnphpflnntewekcnnilsfyrsyleakknfleslkpedweknqyflmlkepktncetivqgwkngfnlprgiftepirkwfmehrknitvaelkrvglvakviplffseeykdsvqpfynylfnvgninkpdeknflnceerrellrkkkdefkkmtdkekeenpsylefqswnkferelrlvrnqdivtwllcmelfnkkkikelnvekiylknintnttkkeknteekngeekiikeknnilnrimpmrlpikvygrenfsknkkkkirrntfftvyieekgtkllkqgnfkalerdrrlgglfsfvkthskaesksntisksrveyelgeyqkarieiikdmlaleetlidkynsldtdnfhnmltgwlklkdepdkasfqndvdlliavrnafshnqypmrnriafaninpfslssantseekglgianqlkdkthktiekiieiekpietke (SEQ ID NO:  599)Prevotella meddkkttdsiryelkdkhfwaaflnlarhnvyitvnhinkileegeinrdgyettlkntintermedia wneikdinkkdrlskliikhfpfleaatyrinptdttkqkeekqaeaqsleslrksffvfATCC 25611 = DSMiyklrdlrnhyshykhskslerpkfeegllekmynifnasirlvkedyqynkdinpdedf 20706khldrteeefnyyftkdnegnitesgllffvslflekkdaiwmqqklrgfkdnrenkkkmNZ_JAEZ01000017.1)tnevfcrsrmllpklrlqstqtqdwilldmlnelircpkslyerlreedrekfrvpieia >WP_036860899.1dedydaeqepfkntivrhqdrfpyfalryfdyneiftnlrfqidlgtyhfsiykkqigdykeshhlthklygferigeftkqnrpdewrkfvktfnsfetskepyipettphyhlenqkigirfrndndkiwpslktnseknekskykldksfqaeaflsvhellpmmfyylllktentdndneietkkkenkndkqekhkieeiienkiteiyalydtfangeiksideleeyckgkdieighlpkqmiailkdehkvmateaerkqeemlvdvqkslesldnqineeienverknsslksgkiaswlvndmmrfqpvqkdnegkpinnskansteyqllqrtlaffgseherlapyfkqtkliessnphpflkdtewekcnnilsfyrsyleakknfleslkpedweknqyflklkepktkpktivqgwkngfnlprgiftepirkwfmkhrenitvaelkrvglvakviplffseeykdsvqpfynyhfnvgninkpdeknflnceerrellrkkkdefkkmtdkekeenpsylefkswnkferelrlvrnqdivtwllcmelfnkkkikelnvekiylknintnttkkeknteekngeeknikeknnilnrimpmrlpikvygrenfsknkkkkirrntfftvyieekgtkllkqgnfkalerdrrlgglfsfvktpskaesksntisklrveyelgeyqkarieiikdmlalektlidkynsldtdnfnkmltdwlelkgepdkasfqndvdlliavrnafshnqypmrnriafaninpfslssantseekglgianqlkdkthktiekiieiekpietke (SEQ ID NO:  600)Prevotella meddkkttdsiryelkdkhfwaaflnlarhnvyitvnhinkileedeinrdgyentlensintermedia wneikdinkkdrlskliikhfpfleattyrqnptdttkqkeekqaeaqsleslkksffvf(NZ_LBGT01000010.1)iyklrdlrnhyshykhskslerpkfeedlqnkmynifdvsiqfvkedykhntdinpkkdf >WP_061868553.1khldrkrkgkfhysfadnegnitesgllffvslflekkdaiwvqkklegfkcsnksyqkmtnevfcrsrmllpklrlestqtqdwilldmlnelircpkslyerlqgvnrkkfyvsfdpadedydaeqepfkntivrhqdrfpyfalryfdynevfanlrfqidlgtyhfsiykkliggqkedrhlthklygferigefdkqnrpdewkaivkdsdtfkkkeekeeekpyisettphyhlenkkigiafknhniwpstqteltnnkrkkynlgtsikaeaflsvhellpmmfyylllktentkndnkvggkketkkqgkhkieaiieskikdiyalydafangeinsedelkeylkgkdikivhlpkqmiailknehkdmaekaeakqekmklatenrlktldkqlkgkiqngkrynsapksgeiaswlvndmmrfqpvqkdengeslnnskansteyqllqrtlaffgseherlapyfkqtkliessnphpflndtewekcsnilsfyrsylkarknfleslkpedweknqyflmlkepktnretivqgwkngfnlprgfftepirkwfmehwksikvddlkrvglvakvtplffsekykdsvqpfynypfnvgdvnkpkeedflhreerielwdkkkdkfkgykakkkfkemtdkekeehrsylefqswnkferelrlvrnqdivtwllctelidklkidelnikelkklrlkdintdtakkeknnilnrvmpmelpvtvykvnkggyiiknkplhtiyikeaetkllkqgnfkalvkdrringlfsfvktpseaesesnpisklrveyelgkyqnarldiiedmlalekklidkynsldtdnfhnmltgwlelkgeakkarfqndvklltavrnafshnqypmydenlfgnierfslsssniieskgldiaaklkeevskaakkigneednkkeket (SEQ ID NO: 601) Prevotellamkmeddkktkestnmldnkhfwaaflnlarhnvyitvnhinkvlelknkkdqdiiidndqintermedia 17dilaikthwekvngdlnkterlrelmtkhfpfletaiytknkedkeevkqekqakaqsfd(CP003502.1)slkhclflfleklqearnyyshykysestkepmlekellkkmynifddniqlvikdyqhn >AFJ07523.1kdinpdedfkhldrteeefnyyfttnkkgnitasgllffvslflekkdaiwmqqklrgfkdnreskkkmthevfcrsrmllpklrlestqtqdwilldmlnelircpkslyerlqgeyrkkfnvpfdsadedydaeqepfkntivrhqdrfpyfalryfdyneiftnlrfqidlgtyhfsiykkliggqkedrhlthklygferigefakqnrtdewkaivkdfdtyetseepyisetaphyhlenqkigirfrndndeiwpslktngennekrkykldkqyqaeaflsvhellpmmfyylllkkeepnndkknasivegfikreirdiyklydafangeinniddlekycedkgipkrhlpkqmvailydehkdmaeeakrkqkemvkdtkkllatlekqtqgeiedggrnirllksgeiarwlvndmmrfqpvqkdnegnpinnskansteyqmlqrslalynkeekptryfrqvnlinssnphpflkwtkweecnnilsfyrsyltkkieflnklkpedweknqyflklkepktnretivqgwkngfnlprgiftepirewfkrhqndseeyekvetldrvglvtkviplffkkedskdkeeylkkdaqkeinncvqpfygfpynvgnihkpdekdflpseerkklwgdkkykfkgykakvkskkltdkekeeyrsylefqswnkferelrlvrnqdivtwllctelidklkveglnveelkklrlkdidtdtakqeknnilnrvmpmqlpvtvyeiddshnivkdrplhtvyieetktkllkqgnfkalvkdrringlfsfvdtssetelksnpiskslveyelgeranarietikdmilleetliekyktlptdnfsdmlngwlegkdeadkarfqndvkllvavrnafshnqypmrnriafaninpfslssadtseekkldianqlkdkthkiikriieiekpietke (SEQ ID NO: 602)Prevotella meddkktkestnmldnkhfwaaflnlarhnvyitvnhinkvlelknkkdqdiiidndqdiintermedia laikthwekvngdlnkterlrelmtkhfpfletaiytknkedkeevkqekqakaqsfdsl(NZ_AP014926.1)khclflfleklqearnyyshykysestkepmlekellkkmynifddniqlvikdyqhnkd >WP_050955369.1inpdedfkhldrteeefnyyfttnkkgnitasgllffvslflekkdaiwmqqklrgfkdnreskkkmthevfcrsrmllpklrlestqtqdwilldmlnelircpkslyerlqgeyrkkfnvpfdsadedydaeqepfkntivrhqdrfpyfalryfdyneiftnlrfqidlgtyhfsiykkliggqkedrhlthklygferigefakqnrtdewkaivkdfdtyetseepyisetaphyhlenqkigirfrndndeiwpslktngennekrkykldkqyqaeaflsvhellpmmfyylllkkeepnndkknasivegfikreirdiyklydafangeinniddlekycedkgipkrhlpkqmvailydehkdmaeeakrkqkemvkdtkkllatlekqtqgeiedggrnirllksgeiarwlvndmmrfqpvqkdnegnpinnskansteyqmlqrslalynkeekptryfrqvnlinssnphpflkwtkweecnnilsfyrsyltkkieflnklkpedweknqyflklkepktnretlvqgwkngfnlprgiftepirewfkrhqndseeyekvetldrvglvtkviplffkkedskdkeeylkkdaqkeinncvqpfygfpynvgnihkpdekdflpseerkklwgdkkykfkgykakvkskkltdkekeeyrsylefqswnkferelrlvrnqdivtwllctelidklkveglnveelkklrlkdidtdtakqeknnilnrvmpmqlpvtvyeiddshnivkdrplhtvyieetktkllkqgnfkalvkdrringlfsfvdtssetelksnpiskslveyelgeranarietikdmllleetliekyktlptdnfsdmlngwlegkdeadkarfqndvkllvavrnafshnqypmrnriafaninpfslssadtseekkldianqlkdkthkiikriieiekpietke (SEQ ID NO: 603)Prevotella meddkkttdsisyelkdkhfwaaflnlarhnvyitvnhinkvlelknkkdqdiiidndqdintermedia ilaikthwekvngdlnkterlrelmtkhfpfletaiysknkedkeevkqekqakaqsfds(AP014598.1)lkhclflfleklqetrnyyshykysestkepmlekellkkmynifddniqlvikdyqhnk BAU18623.1dinpdedfkhldrteedfnyyftrnkkgnitesgllffvslflekkdaiwmqqklrgfkdnreskkkmthevfcrsrmllpklrlestqtqdwilldmlnelircpkslyerlqgedrekfkvpfdpadedydaeqepfkntivrhqdrfpyfalryfdyneiftnlrfqidlgtfhfsiykkliggqkedrhlthklygferigefakqnrpdewkaivkdldtyetsneryisettphyhlenqkigirfrndndeiwpslktngennekskykldkqyqaeaflsvhellpmmfyylllkkeepnndkknasivegfikreirdmyklydafangeinniddlekycedkgipkrhlpkqmvailydehkdmvkeakrkqrkmvkdtekllaalekqtqektedggrnirllksgeiarwlvndmmrfqpvqkdnegnpinnskansteyqmlqrslalynkeekptryfrqvnlinssnphpflkwtkweecnnilsfyrsyltkkieflnklkpedweknqyflklkepktnretlvqgwkngfnlprgiftepirewfkrhqndskeyekvealdrvglvtkviplffkkedskdkeedlkkdaqkeinncvqpfysfpynvgnihkpdekdflhreerielwdkkkdkfkgykakvkskkltdkekeeyrsylefqswnkferelrlvrnqdivtwllctelidklkveglnveelkklrlkdidtdtakqeknnilnrvmpmqlpvtvyeiddshnivkdrplhtvyieetktkllkqgnfkalvkdrringlfsfvdtsseaelksnpiskslveyelgepanarietikdmllleetliekyknlptdnfsdmlngwlegkdeadkarfqndvkllvavrnafshnqypmrnriafaninpfslssadtseekkldianqlkdkthkiikriieiekpietke (SEQ ID NO: 604)Prevotella mkmeddkkttestnmldnkhfwaaflnlarhnvyitvnhinkvlelknkkdqdiiidndqintermedia ZTdilaikthwekvngdlnkterlrelmtkhfpfletaiytknkedkeevkqekqaeaqsle(ATMK01000017.1)slkdclflfleklqearnyyshykysestkepmleegllekmynifddniqlvikdyqhn >KJJ86756.1kdinpdedfkhldrkgqfkysfadnegnitesgllffvslflekkdaiwmqqkltgfkdnreskkkmthevfcrrrmllpklrlestqtqdwilldmlnelircpkslyerlqgeyrkkfnvpfdsadedydaeqepfkntivrhqdrfpyfalryfdyneiftnlrfqidlgtyhfsiykkliggqkedrhlthklygferigefakqnrpdewkalvkdldtyetsneryisettphyhlenqkigirfrngnkeiwpslktngennekskykldkpyqaeaflsvhellpmmfyylllkkeepnndkknasivegfikreirdmyklydafangeinnigdlekycedkgipkrhlpkqmvailydepkdmvkeakrkqkemvkdtkkllatlekqtqeeiedggrnirllksgeiarwlvndmmrfqpvqkdnegnpinnskansteyqmlqrslalynkeekptryfrqvnlinssnphpflkwtkweecnnilsfyrnyltkkieflnklkpedweknqyflklkepktnretlvqgwkngfnlprgiftepirewfkrhqndskeyekvealkrvglvtkviplffkeeyfkedaqkeinncvqpfysfpynvgnihkpdekdflpseerkklwgdkkdkfkgykakvkskkltdkekeeyrsylefqswnkferelrlvrnqdivtwllctelidkmkveglnveelqklrlkdidtdtakqeknnilnrimpmqlpvtvyeiddshnivkdrplhtvyieetktkllkqgnfkalvkdrringlfsfvdtsskaelkdkpisksvveyelgepanarietikdmillektlikkyeklptdnfsdmlngwlegkdesdkarfqndvkllvavrnafshnqypmrnriafaninpfslssadiseekkldianqlkdkthkiikkiieiekpietke (SEQ ID NO:  605)Prevotella meddkkttgsisyelkdkhfwaaflnlarhnvyitinhinklleireidndekvldiktlaurantiaca wqkgnkdlnqkarlrelmtkhfpfletaiytknkedkkevkqekqaeaqsleslkdclflJCM 15754 fldklqearnyyshykysefskepefeegllekmynifgnniqlvindyqhnkdinpdedNZ_BAKF01000019.1)fkhldrkgqfkysfadnegnitesgllffvslflekkdaiwmqqklngfkdnlenkkkmt >WP_025000926.1hevfcrsrilmpklrlestqtqdwilldmlnelircpkslyerlqgddrekfkvpfdpadedynaeqepfkntlirhqdrfpyfvlryfdyneifknlrfqidlgtyhfsiykkliggqkedrhlthklygferigefakqnrpdewkaivkdldtyetsnkryisettphyhlenqkigirfrngnkeiwpslktndennekskykldkqyqaeaflsvhellpmmfyylllkkekpnndeinasivegfikreirnifklydafangeinniddlekycadkgipkrhlpkqmvailydehkdmvkeakrkqkemvkdtkkllatlekqtqkekeddgrnvkllksgeiarwlvndmmrfqpvqkdnegkpinnskansteyqmlqrslalynneekptryfrqvnliesnnphpflkwtkweecnniltfyysyltkkieflnklkpedwkknqyflklkepktnretivqgwkngfnlprgiftepirewfkrhqnnskeyekvealdrvglvtkviplffkeeyfkdkeenfkedtqkeindcvqpfynfpynvgnihkpkekdflhreerielwdkkkdkfkgykekikskkltekdkeefrsylefqswnkferelrlvrnqdivtwllckelidklkidelnieelkklrinnidtdtakkeknnilnrvmpmelpvtvyeiddshkivkdkplhtiyikeaetkllkqgnfkalvkdrringlfsfvktnseaeskrnpisklrveyelgeyqearieliqdmlaleeklinkykdlptnkfsemlnswlegkdeadkarfqndvdfliavrnafshnqypmhnkiefanikpfslytannseekglgianqlkdktkettdkikkiekpietke (SEQ ID NO:  606)Prevotella mendkrleesacytlndkhfwaaflnlarhnvyitvnhinktlelknkknqeiiidndqdpleuritidis ilaikthwakvngdlnktdrlrelmikhfpfleaaiysnnkedkeevkeekqakaqsfksF0068 lkdclflfleklqearnyyshykysesskepefeegllekmyntfdasirlvkedyqynkNZ_AWET01000045.1)didpekdfkhlerkedfnylftdkdnkgkitkngllffvslflekkdaiwmqqkfrgfkd >WP_021584635.1nrgnkekmthevfcrsrmllpkirlestqtqdwilldmlnelircpkslyerlqgayrekfkvpfdsidedydaeqepfrntivrhqdrfpyfalryfdyneifknlrfqidlgtyhfsiykkliggkkedrhlthklygferigeftkqnrpdkwqaiikdldtyetsneryisettphyhlenqkigirfrndnndiwpslktngeknekskynldkpyqaeaflsvhellpmmfyylllkmentdndkednevgtkkkgnknnkqekhkieeiienkikdiyalydaftngeinsidelaegregkdieighlpkqlivilknkskdmaekanrkqkemikdtkkrlatldkqvkgeiedggrnirllksgeiarwlvndmmrfqpvqkdnegkpinnskansteyqmlqrslalynkeekptryfrqvnlikssnphpfledtkweecynilsfyrnylkakikflnklkpedwkknqyflmlkepktnrktivqgwkngfnlprgiftepikewfkrhqndseeykkvealdrvglvakviplffkeeyfkedaqkeinncvqpfysfpynvgnihkpeeknflhceerrklwdkkkdkfkgykakekskkmtdkekeehrsylefqswnkferelrlvrnqdiltwllctklidklkidelnieelqklrlkdidtdtakkeknnilnrvmpmrlpvtvyeidksfnivkdkplhtvyieetgtkllkqgnfkalvkdrringlfsfvktsseaeskskpisklrveyelgayqkaridiikdmlalektlidndenlptnkfsdmlkswlkgkgeankarlqndvgllvavrnafshnqypmynsevfkgmkllslssdipekeglgiakqlkdkiketieriieiekeirn(SEQ ID NO: 607) Prevotellamendkrleestcytlndkhfwaaflnlarhnvyitinhinklleirgidndekvldikal pleuritidiswqkvdkdinqkarlrelmikhfpfleaaiysnnkedkeevkeekqakaqsfkslkdclfl JCM 14110fleklqearnyyshykssesskepefeegllekmyntfgvsirlvkedyqynkdidpekd(NZ_BAJN01000005.1)fkhlerkedfnylftdkdnkgkitkngllffvslflekkdaiwmqqklrgfkdnrgnkek >WP_036931485.1mthevfcrsrmllpkirlestqtqdwilldmlnelircpkslyerlqgayrekfkvpfdsidedydaeqepfrntivrhqdrfpyfalryfdyneifknlrfqidlgtyhfsiykkligdnkedrhlthklygferigefakqkrpnewqalvkdldiyetsnegyisettphyhlenqkigirfknkkdkiwpsletngkenekskynldksfqaeaflsihellpmmfydlllkkeepnndeknasivegfikkeikrmyaiydafaneeinskegleeycknkgfgerhlpkqmiailtnksknmaekakrkqkemikdtkkrlatldkqvkgeiedggrnirllksgeiarwlvndmmrfqsvqkdkegkpinnskansteyqmlqrslalynkeqkptpyfiqvnlikssnphpfleetkweecnnilsfyrsyleakknfleslkpedwkknqyflmlkepktnrktivqgwkngfnlprgiftepikewfkrhqndseeykkvealdrvglvakviplffkeeyfkedaqkeinncvqpfysfpynvgnihkpeeknflhceerrklwdkkkdkfkgykakekskkmtdkekeehrsylefqswnkferelrlvrnqdivtwllctelidklkidelnieelqklrlkdidtdtakkeknnilnrimpmqlpvtvyeidksfnivkdkplhtiyieetgtkllkqgnfkalvkdrringlfsfvktsseaeskskpisklrveyelgayqkaridiikdmlalektlidndenlptnkfsdmlkswlkgkgeankarlqndvdllvairnafshnqypmynsevfkgmkllslssdipekeglgiakqlkdkiketieriieiekeirn (SEQ ID NO: 608)Prevotella falseniimkndnnstkstdytlgdkhfwaaflnlarhnvyitvnhinkvlelknkkdqeiiidndqd DSM 22864 =ilaiktlwgkvdtdinkkdrlrelimkhfpfleaatyqqsstnntkqkeeeqakaqsfes J0415124lkdclflfleklrearnyyshykhsksleepkleekllenmynifdtnvqlvikdyehnkNZ_BAJY01000004.1)dinpeedfkhlgraegefnyyftrnkkgnitesgllffvslflekkdaiwaqtkikgfkd >WP_036884929.1nrenkqkmthevfcrsrmllpklrlestqtqdwilldmlnelircpkslykrlqgekrekfrvpfdpadedydaeqepfkntivrhqdrfpyfalryfdyneiftnlrfqidlgtyhfsiykkqigdkkedrhlthklygferigefakenrpdewkalvkdldtfeesnepyisettphyhlenqkigirnknkkkkktiwpsletkttvnerskynlgksfkaeaflsvhellpmmfyylllnkeepnngkinaskvegiiekkirdiyklygafaneeinneeelkeycegkdiairhlpkqmiailkneykdmakkaedkqkkmikdtkkrlaaldkqvkgevedggrnikplksgriaswlvndmmrfqpvqrdrdgypinnskansteyqllqrtlalfgsererlapyfrqmnligkdnphpflkdtkwkehnnilsfyrsyleakknflgslkpedwkknqyflklkepktnretivqgwkngfnlprgiftepirewfirhqneseeykkvkdfdriglvakviplffkedyqkeiedyvqpfygypfnvgnihnsgegtflnkkereelwkgnktkfkdyktkeknkektnkdkfkkktdeekeefrsyldfqswkkferelrlvrnqdivtwllcmelidklkidelnieelqklrlkdidtdtakkeknnilnrimpmelpvtvyetddsnniikdkplhtiyikeaetkllkqgnfkalvkdrringlfsfvetsseaelkskpiskslveyelgeyqrarveiikdmlrleetligndeklptnkfrqmldkwlehkketddtdlkndvklltevrnafshnqypmrdriafanikpfslssantsneeglgiakklkdktketidriieieeqtatkr (SEQ ID NO: 609)Porphyromonas gulaemteqserpyngtyytledkhfwaaflnlarhnayitlthidrqlayskaditndqdvlsfNZ_JRAT01000012.1)kalwknldndlerksrlrslilkhfsflegaaygkklfeskssgnkssknkeltkkekee >WP_039418912.1lqanalsldnlksilfdflqklkdfrnyyshyrhsgsselplfdgnmlqrlynvfdvsvqrvkrdhehndkvdphrhfnhlvrkgkkdryghndnpsfkhhfvdsegmvteagllffvslflekrdaiwmqkkirgfkggtetyqqmtnevfcrsrislpklkleslrmddwmlldmlnelvrcpkplydrlreddracfrvpvdilpdeddtdgggedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykkmigeqpedrhltrnlygfgriqdfaeehrpeewkrlvrdldyfetgdkpyisqtsphyhiekgkiglrfmpegqhlwpspevgttrtgrskyaqdkrltaeaflsvhelmpmmfyyfllrekyseevsaekvqgrikrviedvyaiydafardeintlkeldacladkgirrghlpkqmiailsgehknmeekvrkklqemiadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdasgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpflhdtrweshtnilsfyrsylrarkaflerigrsdrmenrpflllkepktdrqtivagwksefhlprgifteavrdcliemgydevgsyrevgfmakavplyferacedrvqpfydspfnvgnslkpkkgrflskeeraeewergkerfrdleawshsaarriedafagieyaspgnkkkieqllrdlslweafesklkvradkinlaklkkeileagehpyhdfkswqkferelrlvknqdiitwmmcrdlmeenkvegldtgtlylkdirtnvqeqgslnvinhvkpmrlpvvvyradsrghvhkeeaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgglamegypisklrveyelakyqtarvcafeqtleleeslltryphlpdknfrkmleswsdpllakwpelhgkvrlliavrnafshnqypmydeavfssirkydpsspdaieermglniahrlseevkqaketveriiqa (SEQ ID NO: 610) Porphyromonasmteqserpyngtyytledkhfwaaflnlarhnayitlthidrqlayskaditndqdvlsfsp. COT-052 OH4946kalwknfdndlerksrlrslilkhfsflegaaygkklfeskssgnkssknkeltkkekee(NZ_JQZY01000014.1)lqanalsldnlksilfdflqklkdfrnyyshyrhsesselplfdgnmlqrlynvfdvsvq >WP_039428968.1rvkrdhehndkvdphrhfnhlvrkgkkdryghndnpsfkhhfvdsegmvteagllffvslflekrdaiwmqkkirgfkggtetyqqmtnevfcrsrislpklkleslrtddwmlldmlnelvrcpkplydrlreddracfrvpvdilpdeddtdgggedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykkmigeqpedrhltrnlygfgriqdfaeehrpeewkrlvrdldyfetgdkpyisqttphyhiekgkiglrfvpegqhlwpspevgttrtgrskyaqdkrltaeaflsvhelmpmmfyyfllrekyseevsaekvqgrikrviedvyaiydafardeintlkeldacladkgirrghlpkqmigilsgerkdmeekvrkklqemiadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdtsgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpflhetrweshtnilsfyrsylrarkaflerigrsdrvencpflllkepktdrqtivagwkgefhlprgifteavrdcliemgydevgsyrevgfmakavplyferacedrvqpfydspfnvgnslkpkkgrflskedraeewergkerfrdleawshsaarrikdafagieyaspgnkkkieqllrdlslweafesklkvradkinlaklkkeileagehpyhdfkswqkferelrlvknqdiitwmmcrdlmeenkvegldtgtlylkdirpnvqeqgslnvinrvkpmrlpvvvyradsrghvhkeeaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgglamegypisklrveyelakyqtarvcvfeltlrleesllsryphlpdesfremleswsdpllakwpelhgkvrlliavrnafshnqypmydeavfssirkydpsspdaieermglniahrlseevkqaketveriiqa (SEQ ID NO: 611) Porphyromonas gulaemteqserpyngtyytledkhfwaaflnlarhnayitlthidrqlayskaditndqdvlsf(NZ_JRFD01000046.1)kalwknldndlerksrlrslilkhfsflegaaygkklfeskssgnkssknkeltkkekee >WP_039442171Alqanalsldnlksilfdflqklkdfrnyyshyrhsgsselplfdgnmlqrlynvfdvsvqrvkrdhehndkvdphyhfnhlvrkgkkdryghndnpsfkhhfvdsegmvteagllffvslflekrdaiwmqkkirgfkggtgpyeqmtnevfcrsrislpklkleslrtddwmlldmlnelvrcpkplydrlrekdracfrvpvdilpdeddtdgggedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykkmigeqpedrhltrnlygfgriqdfaeehrpeewkrlvrdldyletgdkpyisqttphyhiekgkiglrfvpegqhlwpspevgttrtgrskcaqdkrltaeaflsvhelmpmmfyyfllrekyseevsaekvqgrikrviedvyaiydafardeintlkeldtcladkgirrghlpkqmitilsgerkdmkekirkklqemiadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdasgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpflhetrweshtnilsfyrsylrarkaflerigrsdrvencpflllkepktdrqtivagwkdefhlprgifteavrdcliemgydevgsyrevgfmakavplyferacedrvqpfydspfnvgnslkpkkgrflskedraeewergmerfrdleawshsaarrikdafagieyaspgnkkkieqllrdlslweafesklkvradkinlaklkkeileagehpyhdfkswqkferelrlvknqdiitwmmcrdlmeenkvegldtgtlylkdirpnvqeqgslnvinrvkpmrlpvvvyradsrghvhkeaplatvyieerntkllkqgnfksfvkdrringlfsfvdtgglamegypisklrveyelakyqtarvcvfeltlrleesllsryphlpdesfremleswsdpllakwpelhgkvrlliavrnafshnqypmydeavfssirkydpsspdaieermglniahrlseevkqaketveriiqa (SEQ ID NO: 612) Porphyromonas gulaemteqserpyngtyytledkhfwaaflnlarhnayitlthidrqlayskaditndqdvlsf(NZ_JRAJ01000010.1)kalwknfdndlerksrlrslilkhfsflegaaygkklfeskssgnkssknkeltkkekee >WP_039431778Alqanalsldnlksilfdflqklkdfrnyyshyrhsesselplfdgnmlqrlynvfdvsvqrvkrdhehndkvdphrhfnhlvrkgkkdryghndnpsfkhhfvdgegmvteagllffvslflekrdaiwmqkkirgfkggtetyqqmtnevfcrsrislpklkleslrtddwmlldmlnelvrcpkplydrlreddracfrvpvdilpdeddtdgggedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykkmigeqpedrhltrnlygfgriqdfaeehrpeewkrlvrdldyfetgdkpyisqtsphyhiekgkiglrfmpegqhlwpspevgttrtgrskyaqdkrltaeaflsvhelmpmmfyyfllrekyseevsaekvqgrikrviedvyaiydafardeintlkeldacladkgirrghlpkqmiailsgehkdmeekirkklqemiadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdtsgkpinnskansteyrmlqralalfggekkrltpyfrqmnitggnnphpflhetrweshtnilsfyrsylrarkaflerigrsdrmenrpflllkepktdrqtivagwksefhlprgifteavrdcliemgydevgsyrevgfmakavplyferacedrvqpfydspfnvgnslkpkkgrflskeeraeewergkerfrdleawshsaarriedafagieyaspgnkkkieqllrdlslweafesklkvradkinlaklkkeileagehpyhdfkswqkferelrlvknqdiitwmmcrdlmeenkvegldtgtlylkdirpnvqeqgslnvinrvkpmrlpvvvyradsrghvhkeeaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgglamegypisklrveyelakyqtarvcvfeltlrleeslltryphlpdesfrkmleswsdpllakwpelhgkvrlliavrnafshnqypmydeavfssirkydpsspdaieermglniahrlseevkqaketveriiqv (SEQ ID NO: 613) Porphyromonas gulaemteqserpyngtyytledkhfwaaflnlarhnayitlthidrqlayskaditndqdvlsf(NZ_KQ040500.1)kalwknfdndlerksrlrslilkhfsflegaaygkklfeskssgnkssknkeltkkekee >WP_046201018Alqanalsldnlksilfdflqklkdfrnyyshyrhsesselplfdgnmlqrlynvfdvsvqrvkrdhehndkvdphrhfnhlvrkgkkdryghndnpsfkhhfvdsegmvteagllffvslflekrdaiwmqkkirgfkggtetyqqmtnevfcrsrislpklkleslrtddwmlldmlnelvrcpkplydrlrekdrarfrvpvdilpdeddtdgggedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykkmigeqpedrhltrnlygfgriqdfaeehrpeewkrlvrdldyfetgdkpyisqttphyhiekgkiglrfmpegqhlwpspevgttrtgrskyaqdkrltaeaflsvhelmpmmfyyfllrekyseevsaekvqgrikrviedvyaiydafardeintlkeldacladkgirrghlpkqmiailsgehkdmeekirkklqemiadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdtsgkpinnskansteyrmlqralalfggekkrltpyfrqmnitggnnphpflhetrweshtnilsfyrsylrarkaflerigrsdrmenrpflllkepktdrqtivagwksefhlprgifteavrdcliemgydevgsyrevgfmakavplyferacedrvqpfydspfnvgnslkpkkgrflskeeraeewergkerfrdleawshsaarriedafagieyaspgnkkkieqllrdlslweafesklkvradkinlaklkkeileagehpyhdfkswqkferelrlvknqdiitwmmcrdlmeenkvegldtgtlylkdirpnvqeqgslnvinrvkpmrlpvvvyradsrghvhkeeaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgglamegypisklrveyelakyqtarvcvfeltlrleeslltryphlpdesfrkmleswsdpllakwpelhgkvrlliavrnafshnqypmydeavfssirkydpsspdaieermglniahrlseevkqaketveriiqv (SEQ ID NO: 614) Porphyromonas gulaemteqserpyngtyytledkhfwaaflnlarhnayitlthidrqlayskaditndqdvlsf(NZ_JRAL01000022.1)kalwknfdndlerksrlrslilkhfsflegaaygkklfeskssgnkssknkeltkkekee >WP_039434803.1lqanalsldnlksilfdflqklkdfrnyyshyrhsgsselplfdgnmlqrlynvfdvsvqrvkidhehndevdphyhfnhlvrkgkkdryghndnpsfkhhfvdgegmvteagllffvslflekrdaiwmqkkirgfkggtetyqqmtnevfcrsrislpklkleslrmddwmlldmlnelvrcpkplydrlreddracfrvpvdilpdeddtdgggedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykkmigeqpedrhltrnlygfgriqdfaeehrpeewkrlvrdldyfetgdkpyisqtsphyhiekgkiglrfmpegqhlwpspevgttrtgrskyaqdkrltaeaflsvhelmpmmfyyfllrekyseevsaervqgrikrviedvyavydafardeintrdeldacladkgirrghlprqmiailsgehkdmeekirkklqemmadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdasgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpflhetrweshtnilsfyrsylrarkaflerigrsdrvenrpflllkepktdrqtivagwkgefhlprgifteavrdcliemghdevasykevgfmakavplyferacedrvqpfydspfnvgnslkpkkgrflskeeraeewergkerfrdleawsysaarriedafagieyaspgnkkkieqllrdlslweafesklkvradrinlaklkkeileagehpyhdfkswqkferelrlvknqdiitwmmcrdlmeenkvegldtgtlylkdirpnvqeqgslnvinrvkpmrlpvvvyradsrghvhkeeaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgglamegypisklrveyelakyqtarvcvfeltlrleeslltryphlpdesfremleswsdpllakwpelhgkvrlliavrnafshnqypmydeavfssirkydpsspdaieermglniahrlseevkqaketveriiqa (SEQ ID NO: 615) Porphyromonas gulaemteqserpyngtyytledkhfwaaflnlarhnayitlthidrqlayskaditndqdvlsf(NZ_JRAI01000002.1)kalwknldndlerksrlrslilkhfsflegaaygkklfeskssgnkssknkeltkkekee >WP_039419792.1lqanalsldnlksilfdflqklkdfrnyyshyrhsgsselplfdgnmlqrlynvfdvsvqrvkrdhehndkvdphrhfnhlvrkgkkdryghndnpsfkhhfvdgegmvteagllffvslflekrdaiwmqkkirgfkggtetyqqmtnevfcrsrislpklkleslrtddwmlldmlnelvrcpkplydrlrekdrarfrvpvdilpdeddtdgggedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykkvigeqpedrhltrnlygfgriqdfaeehrpeewkrlvrdldyfetgdkpyisqttphyhiekgkiglrfvpegqhlwpspevgttrtgrskyaqdkrltaeaflsvhelmpmmfyyfllrekyseevsaekvqgrikrviedvyaiydafardeintrdeldacladkgirrghlpkqmigilsgehknmeekvrkklqemiadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdtsgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpfldetrweshtnilsfyrsylrarkaflerigrsdrvenrpflllkepktdrqtivagwksefhlprgifteavrdcliemgydevgsykevgfmakavplyferackdrvqpfydspfnvgnslkpkkgrflskekraeewesgkerfrlaklkkeileagehpyhdfkswqkferelrlvknqdiitwmmcrdlmeenkvegldtgtlylkdirpnvqeqgslnvinrvkpmrlpvvvyradsrghvhkeeaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgglamegypisklrveyelakyqtarvcvfeltlrleesllsryphlpdesfremleswsdpllakwpelhgkvrlliavrnafshnqypmydeavfssirkydpsspdaieermglniahrlseevkqaketveriiqa (SEQ ID NO: 616)Porphyromonas gulaemteqserpyngtyytledkhfwaaflnlarhnayitlthidrqlayskaditndqdvlsf(NZ_JRAK01000129.1)kalwknfdndlerksrlrslilkhfsflegaaygkklfeskssgnkssknkeltkkekee >WP_039426176.1lqanalsldnlksilfdflqklkdfrnyyshyrhsgsselplfdgnmlqrlynvfdvsvqrvkrdhehndkvdphyhfnhlvrkgkkdryghndnpsfkhhfvdsegmvteagllffvslflekrdaiwmqkkirgfkggtgpyeqmtnevfcrsrislpklkleslrtddwmlldmlnelvrcpkplydrlrekdracfrvpvdilpdeddtdgggedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykkmigeqpedrhltrnlygfgriqdfaeehrpeewkrlvrdldyfetgdkpyisqttphyhiekgkiglrfmpegqhlwpspevgttrtgrskyaqdkrltaeaflsvhelmpmmfyyfllrekyseevsaekvqgrikrvikdvyaiydafardeintlkeldacsadkgirrghlpkqmigilsgehknmeekvrkklqemiadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdtsgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpfldetrweshtnilsfyrsylrarkaflerigrsdrvenrpflllkepkndrqtivagwksefhlprgifteavrdcliemgydevgsykevgfmakavplyferackdrvqpfydspfnvgnslkpkkgrflskekraeewesgkerfrlaklkkeileakehpyhdfkswqkferelrlvknqdiitwmmcrdlmeenkvegldtgtlylkdirtdvheqgslnvinrvkpmrlpvvvyradsrghvhkeqaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgglamegypisklrveyelakyqtarvcafeqtleleeslltryphlpdenfremleswsdpllgkwpdlhgkvrlliavrnafshnqypmydeavfssirkydpsspdaieermglniahrlseevkqaketveriiqa (SEQ ID NO: 617)Porphyromonas gulaemteqserpyngtyytledkhfwaaflnlarhnayitlthidrqlayskaditndedilff(NZ_KN294104.1)kgqwknldndlerksrlrslilkhfsflegaaygkkffeskssgnkssknkeltkkekee >WP_039437199.1lqanalsldnlksilfdflqklkdfrnyyshyrhsgsselplfdgnmlqrlynvfdvsvqrvkrdhehndevdphyhfnhlvrkgkkdryghndnpsfkhhfvdgegmvteagllffvslflekrdaiwmqkkirgfkggtepyeqmtnevfcrsrislpklkleslrtddwmlldmlnelvrcpkplydrlrekdracfrvpvdilpdeddtdgggedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykkmigeqpedrhltrnlygfgriqdfaeehrpeewkrlvrdldyfetgdkpyisqttphyhiekgkiglrfvpegqhlwpspevgttrtgrskyaqdkrltaeaflsvhelmpmmfyyfllrekyseevsaekvqgrikrviedvyaiydafardeintlkeldacladkgirrghlpkqmigilsgerkdmeekvrkklqemiadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdtsgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpflhetrweshtnilsfyrsylrarkaflerigrsdrvencpflllkepktdrqtivagwkgefhlprgifteavrdcliemgydevgsyrevgfmakavplyferacedrvqpfydspfnvgnslkpkkgrflskekraeewesgkerfrlaklkkeileagehpyhdfkswqkferelrlvknqdiitwmmcrdlmeenkvegldtgtlylkdirpnvqeqgslnvinrvkpmrlpvvvyradsrghvhkeeaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgalamegypisklrveyelakyqtarvcafeqtleleeslltryphlpdesfremleswsdplltkwpelhgkvrlliavrnafshnqypmydeavfssiwkydpsspdaieermglniahrlseevkqaketieriiqa (SEQ ID NO: 618) Porphyromonasmtegnekpyngtyytledkhfwaaffnlarhnayitlahidrqlayskaditndedilffgingivalis TDC60kgqwknldndlerkarlrslilkhfsflegaaygkklfesqssgnkssknkeltkkekee(NC_015571.1)lqanalsldnlksilfdflqklkdfrnyyshyrhpesselplfdgnmlqrlynvfdvsvq >WP_013816155.1rvkrdhehndkvdphrhfnhlvrkgkkdrygnndnpffkhhfvdregtvteagllffvslflekrdaiwmqkkirgfkggtetyqqmtnevfcrsrislpklkleslrtddwmlldmlnelvrcpkslydrlreedrarfrvpvdilsdeedtdgaeedpfkntivrhqdrfpyfalryfdlkkvftslrfqidlgtyhfaiykknigeqpedrhltrnlygfgriqdfaeehrpeewkrlvrdldyfetgdkpyitqttphyhiekgkiglrfvpegqhlwpspevgatrtgrskyaqdkrftaeaflsahelmpmmfyyfllrekyseeasaervqgrikrviedvyavydafardeintrdeldacladkgirrghlprqmigilsgehkdmeekirkklqemmadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdtsgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpflhetrweshtnilsfyrsylkarkaflqsigrsdrvenhrflllkepktdrqtivagwkgefhlprgifteavrdcliemgldevgsykevgfmakavplyferackdwvqpfynypfnvgnslkpkkgrflskekraeewesgkerfrlaklkkeileakehpyldfkswqkferelrlvknqdiitwmicgdlmeenkvegldtgtlylkdirtdvqeqgslnvinrvkpmrlpvvvyradsrghvhkeqaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgalamegypisklrveyelakyqtarvcafeqtleleeslltrcphlpdknfrkmleswsdplldkwpdlhrkvrlliavrnafshnqypmydeavfssirkydpsfpdaieermglniahrlseevkqaketveriiqa (SEQ ID NO: 619) Porphyromonasmtegnerpyngtyytledkhfwaaffnlarhnayitlahidrqlayskaditndedilff gingivaliskgqwknldndlerkarlrslilkhfsflegaaygkklfesqssgnksskkkeltkkekee ATCC 33277lqanalsldnlksilfdflqklkdfrnyyshyrhpesselplfdgnmlqrlynvfdvsvq(NC_010729.1)rvkrdhehndkvdphrhfnhlvrkgkkdrygnndnpffkhhfvdreekvteagllffvsl >WP_012458414.1flekrdaiwmqkkirgfkggtetyqqmtnevfcrsrislpklkleslrtddwmlldmlnelvrcpkslydrlreedrarfrvpvdilsdeddtdgteedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykknigeqpedrhltrnlygfgriqdfaeehrpeewkrlvrdldyfetgdkpyitqttphyhiekgkiglrfvpegqhlwpspevgatrtgrskyaqdkrltaeaflsvhelmpmmfyyfllrekysdeasaervqgrikrviedvyavydafargeintrdeldacladkgirrghlprqmigilsgehkdmeekvrkklqemivdtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdtsgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpflhetrweshtnilsfyrsylkarkaflqsigrsdrvenhrflllkepktdrqtivagwkgefhlprgifteavrdcliemgldevgsykevgfmakavplyferackdrvqpfydypfnvgnslkpkkgrflskekraeewesgkerfrlaklkkeileakehpyldfkswqkferelrlvknqdiitwmicrdlmeenkvegldtgtlylkdirtdvqeqgninvinrvkpmrlpvvvyradsrghvhkeqaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgalamegypisklrveyelakyqtarvcafeqtleleeslltryphlpdknfrkmleswsdplldkwpdlhgnvrlliavrnafshnqypmydeavfssirkydpsspdaieermglniahrlseevkqakemaeriiqa (SEQ ID NO: 620) Porphyromonasmtegnekpyngtyytlkdkhfwaaffnlarhnayitlthidrqlayskaditndedilff gingivaliskgqwknldndlerkarlrslilkhfsflegaaygkklfesqssgnksskkkeltkkekee A7A1-28lqanalsldnlksilfdflqklkdfrnyyshyrhpesselpmfdgnmlqrlynvfdvsvq(NZCP013131.1)rvkrdhehndkvdphrhfnhlvrkgkkdrcgnndnpffkhhfvdregkvteagllffvsl >WP_058019250.1flekrdaiwmqkkirgfkggtetyqqmtnevfcrsrislpklkleslrtddwmlldmlnelvrcpkslydrlreedracfrvpvdilsdeddtdgaeedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykknigeqpedrhltrnlygfgriqdfaeehrpeewkrlvrdldcfetgdkpyitqttphyhiekgkiglrfvpegghlwpspevgatrtgrskyaqdkrftaeaflsvhelmpmmfyyfllrekyseevsaervqgrikrviedvyavydafardeintrdeldacladkgirrghlprqmiailsqkhkdmeekvrkklqemiadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdtsgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpflhetrweshtnilsfyrsylkarkaflqsigrsdrvenhrflllkepktdrqtivagwkgefhlprgifteavrdcliemgldevgsykevgfmakavplyferackdrvqpfydypfnvgnslkpkkgrflskekraeewesgkerfrdleawshsaarriedafagienasrenkkkieqllqdlslwetfesklkvkadkiniaklkkeileakehpyldfkswqkferelrlvknqdiitwmmcrdlmeenkvegldtgtlylkdirtdvqeqgslnvinhvkpmrlpvvvyradsrghvhkeqaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgalamegypisklrveyelakyqtarvcafeqtleleeslltryphlpdenfrkmleswsdplldkwpdlhrkvrlliavrnafshnqypmydeavfssirkydpsspdaieermglniahrlseevkqakemaeriiqa (SEQ ID NO: 621) Porphyromonasmtegnekpyngtyytledkhfwaaffnlarhnayitlthidrqlayskaditndedilff gingivaliskgqwknldndlerkarlrslilkhfsflegaaygkklfesqssgnksskkkeltkkekee JCVI SC001lqanalsldnlksilfdflqklkdfrnyyshyrhpesselplfdgnmlqrlynvfdvsvqAPMB01000175.1)rvkrdhehndkvdphrhfnhlvrkgkkdrcgnndnpffkhhfvdreekvteagllffvsl >EOA10535.1flekrdaiwmqkkirgfkggtetyqqmtnevfcrsrislpklkleslrtddwmlldmlnelvrcpkslydrlreedrarfrvpvdilsdeddtdgteedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykknigeqpedrhltrnlygfgriqdfaeehrpeewkrlvrdldyfetgdkpyitqttphyhiekgkiglrfvpegqllwpspevgatrtgrskyaqdkrftaeaflsvhelmpmmfyyfllrekyseeasaervqgrikrviedvyavydafargeidtldrldacladkgirrghlprqmiailsgehkdmeekvrkklqemiadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdtsgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpflhetrweshtnilsfyrsylkarkaflqsigrsdrvenhrflllkepktdrqtivagwkgefhlprgifteavrdcliemgldevgsykevgfmakavplyferackdrvqpfydypfnvgnslkpkkgrflskekraeewesgkerfrdleawshsaarriedafagienasrenkkkieqllqdlslwetfesklkvkadkiniaklkkeileakehpyldfkswqkferelrlvknqdiitwmmcrdlmeenkvegldtgtlylkdirtdvheqgslnvinrvkpmrlpvvvyradsrghvhkeqaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgalamegypisklrveyelakyqtarvcafeqtleleeslltryphlpdknfrkmleswsdplldkwpdlhgnvrlliavrnafshnqypmydetlfssirkydpsspdaieermglniahrlseevkqakemveriiqa (SEQ ID NO: 622) Porphyromonasmtegnekpyngtyytledkhfwaaffnlarhnayitlahidrqlayskaditndedilffgingivalis W50kgqwknldndlerkarlrslilkhfsflegaaygkklfesqssgnksskkkeltkkekee(NZ_AJZS01000051.1)lqanalsldnlksilfdflqklkdfrnyyshyrhpesselplfdgnmlqrlynvfdvsvq >WP_005874195.1rvkrdhehndkvdphrhfnhlvrkgkkdkygnndnpffkhhfvdreekvteagllffvslflekrdaiwmqkkirgfkggteayqqmtnevfcrsrislpklkleslrtddwmlldmlnelvrcpkslydrlreedrarfrvpvdilsdeddtdgteedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykknigeqpedrhltrnlygfgriqdfaeehrpeewkrlvrdldyfetgdkpyitqttphyhiekgkiglrfvpegqllwpspevgatrtgrskyaqdkrftaeaflsvhelmpmmfyyfllrekyseeasaekvqgrikrviedvyavydafardeintrdeldacladkgirrghlprqmiailsgehkdmeekvrkklqemiadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdtsgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpflhetrweshtnilsfyrsylkarkaflqsigrsdreenhrflllkepktdrqtivagwksefhlprgifteavrdcliemgydevgsykevgfmakavplyferackdrvqpfydypfnvgnslkpkkgrflskekraeewesgkerfrdleawshsaarriedafvgieyaswenkkkieqllqdlslwetfesklkvkadkiniaklkkeileakehpyhdfkswqkferelrlvknqdiitwmmcrdlmeenkvegldtgtlylkdirtdvqeqgslnvinhvkpmrlpvvvyradsrghvhkeeaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgalamegypisklrveyelakyqtarvcafeqtleleeslltryphlpdesfremleswsdplldkwpdlqrevrlliavrnafshnqypmydetifssirkydpssldaieermglniahrlseevklakemveriiqa (SEQ ID NO: 623) Porphyromonasmtegnekpyngtyytledkhfwaaffnlarhnayitlahidrqlayskaditndedilff gingivaliskgqwknldndlerkarlrslilkhfsflegaaygkklfesqssgnksskkkeltkkekee(NZ_CP011995.1)lqanalsldnlksilfdflqklkdfrnyyshyrhpesselplfdgnmlqrlynvfdvsvq >WP_052912312.1rvkrdhehndkvdphrhfnhlvrkgkkdkygnndnpffkhhfvdreekvteagllffvslflekrdaiwmqkkirgfkggteayqqmtnevfcrsrislpklkleslrtddwmlldmlnelvrcpkllydrlreedrarfrvpvdilsdeddtdgteedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykknigeqpedrhltrnlygfgriqdfaeehrpeewkrlvrdldyfetgdkpyitqttphyhiekgkiglrfvpegqllwpspevgatrtgrskyaqdkrftaeaflsvhelmpmmfyyfllrekyseeasaekvqgrikrviedvyavydafardeintrdeldacladkgirrghlprqmiailsgehkdmeekvrkklqemiadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdtsgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpflhetrweshtnilsfyrsylkarkaflqsigrsdreenhrflllkepktdrqtivagwksefhlprgifteavrdcliemgydevgsykevgfmakavplyferackdrvqpfydypfnvgnslkpkkgrflskekraeewesgkerfrdleawshsaarriedafvgieyaswenkkkieqllqdlslwetfesklkvkadkiniaklkkeileakehpyhdfkswqkferelrlvknqdiitwmmcrdlmeenkvegldtgtlylkdirtdvqeqgslnvinhvkpmrlpvvvyradsrghvhkeeaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgalamegypisklrveyelakyqtarvcafeqtleleeslltryphlpdesfremleswsdplldkwpdlqrevrlliavrnafshnqypmydetifssirkydpssldaieermglniahrlseevklakemveriiqa (SEQ ID NO: 624) Porphyromonasmtegnekpyngtyytledkhfwaaflnlarhnayitlahidrqlayskaditndedilffgingivalis AJW4kgqwknldndlerkarlrslilkhfsflegaaygkklfesqssgnksskkkelskkekee(NZ_CP011996.1)lqanalsldnlksilfdflqklkdfrnyyshyrhpesselplfdgnmlqrlynvfdvsvq >WP_053444417.1rvkrdhehndkvdphrhfnhlvrkgkkdkygnndnpffkhhfvdregtvteagllffvslflekrdaiwmqkkirgfkggteayqqmtnevfcrsrislpklkleslrtddwmlldmlnelvrcpkslydrlreedrarfrvpvdilsdeddtdgteedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykknigeqpedrhltrnlygfgriqdfaeehrpeewkrlvrdldyfetgdkpyitqttphyhiekgkiglrfvpegqhlwpspevgatrtgrskyaqdkrltaeaflsvhelmpmmfyyfllrekyseevsaekvqgrikrviedvyavydafardeintrdeldacladkgirrghlprqmiailsgehkdmeekvrkklqemiadtdhrldmldrqtdrkirigrknaglpksgvvadwlvrdmmrfqpvakdtsgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpflhetrweshtnilsfyrsylearkaflqsigrsdrvenhrflllkepktdrqtivagwkgefhlprgifteavrdcliemgydevgsykevgfmakavplyferaskdrvqpfydypfnvgnslkpkkgrflskekraeewesgkerfrlaklkkeileakehpyhdfkswqkferelrlvknqdiitwmmcrdlmeenkvegldtgtlylkdirtdvqeqgslnvinrvkpmrlpvvvyradsrghvhkeqaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgalamegypisklrveyelakyqtarvcafeqtleleeslltryphlpdknfrkmleswsdplldkwpdlhgnvrlliavrnafshnqypmydetlfssirkydpsspdaieermglniahrlseevkqakemveriiqa (SEQ ID NO: 625) Porphyromonasmtegnerpyngtyytledkhfwaaffnlarhnayitlahidrqlayskaditndedilff gingivaliskgqwknldndlerkarlrslilkhfsflegaaygkklfesqssgnksskkkeltkkekee(NZ_CP007756.1)lqanalsldnlksilfdflqklkdfrnyyshyrhpesselplfdgnmlqrlynvfdvsvq >WP_039417390.1rvkrdhehndkvdphrhfnhlvrkgkkdrygnndnpffkhhfvdregtvteagllffvslflekrdaiwmqkkirgfkggteayqqmtnevfcrsrislpklkleslrtddwmlldmlnelvrcpkslydrlreedrarfrvpidilsdeddtdgteedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykknigeqpedrhltrnlygfgriqdfaeehrpeewkrlvrdldyfetgdkpyitqttphyhiekgkiglrfvpegqhlwpspevgatrtgrskyaqdkrltaeaflsvhelmpmmfyyfllrekyseevsaekvqgrikrviedvyavydafargeidtldrldacladkgirrghlprqmiailsgehkdmeekvrkklqemiadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdtsgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpflhetrweshtnilsfyrsylkarkaflqsigrsdreenhrflllkepktdrqtivagwksefhlprgifteavrdcliemgydevgsykevgfmakavplyferackdrvqpfydypfnvgnslkpkkgrflskekraeewesgkerfrlaklkkeileakehpyldfkswqkferelrlvknqdiitwmmcrdlmeenkvegldtgtlylkdirtdvheqgslnvinrvkpmrlpvvvyradsrghvhkeqaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgalamegypisklrveyelakyqtarvcafeqtleleeslltryphlpdknfrkmleswsdplldkwpdlhrkvrlliavrnafshnqypmydeavfssirkydpsspdaieermglniahrlseevkqakemaeriiqv (SEQ ID NO: 626) Porphyromonasmtegnerpyngtyytledkhfwaaffnlarhnayitlthidrqlayskaditndedilff gingivaliskgqwknldndlerkarlrslilkhfsflegaaygkklfenkssgnksskkkeltkkekee(NZ_LOEL01000001.1)lqanalsldnlksilfdflqklkdfrnyyshyrhpesselplfdgnmlqrlynvfdvsvq >WP_061156470.1rvkrdhehndkvdphrhfnhlvrkgkkdrcgnndnpffkhhfvdregkvteagllffvslflekrdaiwmqkkirgfkggteayqqmtnevfcrsrislpklkleslrtddwmlldmlnelvrcpkslydrlreedrarfrvpvdilsdeddtdgteedpfkntivrhqdrfpyfalryfdlkkvftslrfhidlgtyhfaiykknigeqpedrhltrnlygfgriqdfaeehrpeewkrlvrdldyfetgdkpyitqttphyhiekgkiglrfvpegqhlwpspevgatrtgrskyaqdkrltaeaflsvhelmpmmfyyfllrekyseevsaekvqgrikrviedvyavydafargeidtldrldacladkgirrghlprqmiailsgehkdmeekvrkklqemiadtdhrldmldrqtdrkirigrknaglpksgviadwlvrdmmrfqpvakdtsgkpinnskansteyrmlqralalfggekerltpyfrqmnitggnnphpflhetrweshtnilsfyrsylkarkaflqsigrsdreenhrflllkepktdrqtivagwksefhlprgifteavrdcliemgydevgsykevgfmakavplyferackdrvqpfydypfnvgnslkpkkgrflskekraeewesgkerfrlaklkkeileakehpyldfkswqkferelrlvknqdiitwmmcrdlmeenkvegldtgtlylkdirtevqeqgslnvinrvkpmrlpvvvyradsrghvhkeqaplatvyieerdtkllkqgnfksfvkdrringlfsfvdtgglamegypisklrveyelakyqtarvcafeqtleleeslltrcphlpdknfrkmleswsdplldkwpdlqrevwlliavrnafshnqypmydeavfssirkydpsspdaieermglniahrlseevkqakemaeriiqa (SEQ ID NO: 627)

TABLE 14 Bacteroidetes mengtqkgkgiyyyytknedkhyfgsflnl bacteriumannniegiieefrirlslkdeknikeiinn GWA2_31_9 yftdkksytdwerginilkeylpvidyldl(hypothetical aitdkefekidlkgketakrkyfrtnfsll proteinidtiidlrnfythyfhkpisinpdvakfld A2033_10205)knllnveldikkgkmktdktkgalkdgldk >OFX18020 elkklielkkaelkekkiktwnitenvegavyndafnhmvyknnagvtilkdyhksilpd dkidselklnfsisglvfllsmflskkeiegfksnlegfkgkvigengeyeiskfnnslk ymathwifsyltfkglkgrvkntfdketllmgmidelnkvphevygtlskeggnefledi neyvgdneenkksmensivvhpvirkryddkfnyfairfldefanfptlkffvtagnfvh dkrekgiggsmltsdrmikekinvfgklteiakyksdyfsnentletsewelfpnpsyll ignnipvhidlihnteeakgegiaidrikettnpakkrntrkskeeiikiiyqknknikY gdptallssnelpaliyellvnkksgkeleniivekivngyktiagfekggnlsnslitk klkksepnedkinaekiilainreleitenklniiknnraefrtgakrkhifyskelgge atwiaydlkrfmpeasrkewkgfhhselgkflafydrnkndakallnmfwnfdndglign dlnsafrefhfdkfyekylikrdeilegfksfisnfkdepkllkkgikdiyrvfdkryyi ikstnagkegllskpielprgifdnkptyiegvkvesnsalfadwygytysdkhefgsfy dmprdykegfekfelnniksignkknlnksdkfiyfrykqdlkikgiksgdlfiklmvde lfnvvfknnielnlkklygtsderfkngliadvgknrekgdtsdnkmnenfiwnmtipls lenggieepkvklkdigkfrkletddkviglleydkskvwkkleiedelenmpnsyerir rekllkgigefehfllekekfdginhpkhfegdlnpnfktyvingvlrknsklnyteidk lldlehisikdietsakeihlayflihvrnkfghnglpkleafelmkkyykknneetyae yfhkvssqivnefknslekhs(SEQ ID NO: 628)Chryseobacterium mektgtglgiyydhtklqdkyffggffnla jejuensegnnidnvikafiikffperkdkdiniagfl (hypotheticaldiefkdndadsdfqkknkflrihfpvigfl protein tsdndkagfkkkfalllktiselrnfythySAMN05421542_0666) yhksiefpselfellddifvkttseikklk >SDI27289.1kkddktggllnknlseeydirygggierlk elkaqgkrvsltdetairngvfnaafnhliyrdgenvkpsrlyqssysepdpaengisls qnsilfllsmflerketedlksrvkgfkakiikqgeegisglkfmathwvfsylcfkgik qklstefheetlliqiidelskvpdevysafdsktkekfledineymkegnadlsledsk vihpvirkryenkfnyfairfldeylsstslkfqvhvgnyvhdrrvkhingtgfqteriv kdrikvfgrlsnisnlkadyikeqlelpndsngweifpnpsyifidnnvpihvladeatk kgielfkdkrrkeqpeelqkrkgkiskynivsmiykeakgkdklrideplallslneipa llyqilekgatpkdieliiknklterfekiknydpetpapasqiskrlrnnttakgqeal naeklslliereientetklssieekrlkakkeqrrntpqrsifsnsdlgriaawladdi krfmpaeqrknwkgyqhsqlqqslayfekrpqeaflllkegwdtsdgssywnnwvmnsfl ennhfekfyknylmkrvkyfselagnikqhthntkflrkfikqqmpadlfpkrhyilkdl eteknkvlskplvfsrglfdnnptfikgvkvtenpelfaewysygyktehvfqhfygwer dynelldselqkgnsfaknsiyynresqldliklkqdlkikkikiqdlflkriaeklfen vfnypttlsldefyltqeeraekerialaqslreegdnspniikddfiwsktiafrskqi yepaiklkdigkfnrfvlddeeskaskllsydknkiwnkeqlerelsigensyevirrek lfkeignlelqilsnwswdginhprefemedqkntrhpnfkmylvngilrkninlykede dfwleslkendfktlpsevletksemvqllflvilirnqfahnqlpeiqfynfirknype iqnntvaelylnliklavqklkdns (SEQID NO: 629) Chryseobacterium mntrvtgmgvsydhtkkedkhffggflnlacarnipullonun qdnitavikafcikfdknpmssvqfaescf (hypotheticaltdkdsdtdfqnkvryvrthlpvigylnygg protein drntfrqklstllkavdslrnfythyyhspSAMN05444360_11366) lalstelfelldtvfasvavevkqhkmkdd >SHM52812.1ktrqllskslaeeldirykqqlerlkelke qgknidlrdeagirngvinaafnhliykegeiakptlsyssfyygadsaengitisqsgl lfllsmflgkkeiedlksrirgfkakivrdgeenisglkfmathwifsylsfkgmkgrls tdfheetlliqiidelskvpdevyhdfdtatrekfvedineyiregnedfslgdstiihp virkryenkfnyfavrfldefikfpslrfqvhlgnfvhdrrikdihgtgfqtervvkdri kvfgklseisslkteyiekeldldsdtgweifpnpsyvfidnnipiyistnktfkngsse fiklrrkekpeemkmrgedkkekrdiasmignagslnsktplamlslnempallyeilvk kttpeeieliikekldshfeniknydpekplpasqiskrlrnnttdkgkkvinpeklihl inkeidateakfallaknrkelkekfrgkplrqtifsnmelgreatwladdikrfmpdil rknwkgyqhnqlqqslaffnsrpkeaftilqdgwdfadgssfwngwiinsfvknrsfeyf yeayfegrkeyfsslaenikqhtsnhrnlrrfidqqmpkglfenrhyllenleteknkil skplvfprglfdtkptfikgikvdeqpelfaewyqygystehvfqnfygwerdyndlles elekdndfsknsihysrtsqleliklkqdlkikkikiqdlflkliaghifenifkypasf sldelyltqeerinkeqealiqsqrkegdhsdniikdnfigsktvtyeskqisepnvklk digkfnrfllddkvktllsynedkvwnkndldlelsigensyevirreklfkkiqnfelq tltdwpwngtdhpeefgttdnkgvnhpnfkmyvvngilrkhtdwfkegednwlenlneth fknlsfqeletksksiqtafliimirnqfahnqlpavqffefiqkkypeiggsttselyl nfinlavvellellek (SEQ ID NO: 630)Chryseobacterium metqilgngisydhtktedkhffggflnta ureilyticumqnnidllikayiskfessprklnsvqfpdv (hypotheticalcfkkndsdadfqhklqfirkhlpviqylky protein ggnrevlkekfrlllqavdslrnfythfyhSAMN05421786_1011119) kpiqlpnelltlldtifgeignevrqnkmk >SIS70481.1ddktrhllkknlseeldfryqeqlerlrkl ksegkkvdlrdteairngvinaafnhlifkdaedfkptvsyssyyydsdtaengisisqs gllfllsmflgrremedlksrvrgfkariikheeqhvsglkfmathwvfsefcfkgiktr lnadyheetlliglidelskvpdelyrsfdvatrerfiedineyirdgkedkslieskiv hpvirkryeskfnyfairfldefvnfptlrfqvhagnyvhdrriksiegtgfkterlvkd rikvfgklstisslkaeylakavnitddtgwellphpsyvfidnnipihltvdpsfkngv keyqekrklqkpeemknrqggdkmhkpaisskigkskdinpespvallsmneipallyei lvkkaspeeveakirqkltavferirdydpkvplpasqvskrlrnntdtlsynkeklvel ankeveqterklalitknrrecrekvkgkfkrqkvfknaelgteatwlandikrfmpeeq kknwkgyqhsqlqqslaffesrpgearsllqagwdfsdgssfwngwvmnsfardntfdgf yesylngrmkyflrladniaqqsstnklisnfikqqmpkglfdrrlymledlateknkil skplifprgifddkptfkkgvqvseepeafadwysygydvkhkfqefyawdrdyeellre elekdtaftknsihysresqiellakkqdlkvkkvriqdlylklmaeflfenvfghelal pldqfyltqeerlkqeqeaivqsqrpkgddspnivkenfiwsktipfksgrvfepnvklk digkfrnlltdekvdillsynnteigkqvieneliigagsyefirreqlfkeiqqmkrls lrsvrgmgvpirinlk (SEQ ID NO: 631)Sinomicrobium mestttlglhlkyqhdlfedkhyfgggvnl oceaniavgniesifqafaerygiqnplrkngvpai >WP_072319476.1nnifhdnisisnykeylkflkqylpvvgfl eksneinifefredfeilinaiyklrhfythyyhspikledrfytclnelfvavaiqvkk hkmksdktrqllnknlhqllqqlieqkreklkdkkaegekvsldtksienavindafvhl ldkdenirinyssrlsediitkngitlsisgllfllslflqrkeaedlrsriegfkgkgn elrfmathwvfsylnvkrikhrintdfqketlligiadelskvpdevyktldhenrskfl edineyiregnedaslnestvvhgvirkryenkfhylvlryldefvdfpslrfqvhlgny ihdrrdkvidgtnfitnrvikepikvfgklshvsklksdymeslsrehkngwdvfpnpsy nfvghnipifinlrsasskgkelyrdlmkiksekkkksreegipmerrdgkptkieisnq idrnikdnnfkdiypgeplamlslnelpallfellrrpsitpqdiedrmveklyerfqii rdykpgdglstskiskklrkadnstrldgkkllraiqtetrnareklhtleenkalqknr krrtvyttreqgreaswlaqdlkrfmpiasrkewrgyhhsqlqqilafydqnpkqplell eqfwdlkedtyvwnswihkslsqhngfvpmyegylkgrlgyykklesdiigfleehkvlk ryytqqhlnvifrerlyfiktetkqklellarplvfprgifddkptfvqdkkvvdhpelf adwyvysykddhsfqefyhykrdyneifetelswdidfkdnkrqlnpseqmdlfrmkwdl kikkikiqdiflkivaediylkifghkiplslsdfyisrgerltldeqavaqsmrlpgdt ksenqikesnlwqttvpyekeqirepkikldigkfkyflqqqkvinllkydpqhvwtkae leeelyigkhsyevvrremllqkchqlekhilegfrfdgsnhpreleggnhpnfkmyivn giltkrgeleieaenwwlelgnsknsldkvevelltmktipeqkafllilirnkfahnql padnyfhyasnlmnlkksdtyslfwftvadtivqefmsl (SEQ ID NO: 632) Reichenbachiellamktnpliassgekpnykkfntesdksfkki agariperforansfqnkgsiapiaekacknfeikskspvnrdg >WP073124441.1rlhyfsvghafknidsknvfryeldesqmd mkptqflalqkeffdfqgalngllkhirnvnshyvhtfekleiqsinqklitflieafel avihsylneeelsyeaykddpqsgqklvqflcdkfypnkeheveerktilaknkrgaleh sllfievtsdidwklfekhkvftisngkylfhaclfllslflykseangliskikgfkrn ddnqyrskrqiftffskkftsqdvnseeqhlvkfrdviqylnhypsawnkhlelksgypq mtdklmryiveaelyrsfpdqtdnhrfllfaireffgqscldtwtgntpinfsnqeqkgf syeintsaeikdietklkalvlkgpinfkekkeqnrlekdlrrekkeqptnrvkeklltr ighnmlyvsygrnqdrfmdfaarflaetdyfgkdakfkmyqfytsdeqrdhlkeqkkelp kkefeklkyhqsklvdyftyaeqqarypdwdtpfvvennaiqikvtlfngakkivsvqrn lmlylledalysekrenagkglisgyfvhhqkelkdqldileketeisreqkrefkkllp krilhryspaqindttewnpmevileeakaqeqryqlllekailhqteedflkrnkgkqf klrfvrkawhlmylkelymnkvaehghhksfhitkeefndfcrwmfafdevpkykeylcd yfsqkgffnnaefkdliesstslndlyektkqrfegwskdltkqsdenkyllanyesmlk ddmlyvnishfisyleskgkinrnahghiaykalnnvphlieeyyykdrlapeeykshgk lynklktvkledallyemamhylslepalvpkvktkvkdilssniafdikdaaghhlyhl lipfhkidsfvalinhqsqqekdpdktsflakiqpylekvknskdlkavyhyykdtphtl ryedlnmihshivsgsvqftkvalkleeyfiakksitlqiargisyseiadlsnyftdev rntafhfdvpetaysmilqgiesefldreikpqkpkslselstqqvsvctafletlhnnl fdrkddkkerlskareryfeqin(SEQ ID NO: 633)

* * *

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1-50. (canceled)
 51. A non-naturally occurring or engineered compositioncomprising; i) a Type VI-B Cas protein optimized for activity in amammalian cell, and ii) one or more nucleic acid components, the one ormore nucleic acid components capable of forming a complex with the TypeVI-B Cas protein and comprising a guide sequence capable of directingsite specific binding of the complex to a target RNA polynucleotidesequence.
 52. The composition of claim 51, wherein the Type VI-B Casprotein is fused to one or more localization signals.
 53. Thecomposition of claim 52, wherein the localization signal is a nuclearlocalization signal (NLS) or a nuclear export signal (NES)
 54. Thecomposition of claim 51, wherein the Type VI-B Cas protein comprises twoHEPN domains.
 55. The composition of claim 51, wherein the Type VI-B Casprotein is associated with one or more functional domains.
 56. Thecomposition of claim 55, wherein the functional domain modifiestranscription of translation of the target sequence.
 57. The compositionof claim 56, wherein the functional domain comprises a cytosine oradenosine deaminase.
 58. The composition of claim 51, wherein thenucleic acid component comprises a dual direct repeat sequence.
 59. Thecomposition of claim 51, wherein the Type VI-B Cas protein is selectedfrom the group consisting of Porphyromonas gulae Cas13b (accessionnumber WP_039434803), Prevotella sp. P5-125 Cas13b (accession numberWP_044065294), Porphyromonas gingivalis Cas13b (accession numberWP_053444417), Porphyromonas sp. COT-052 OH4946 Cas13b (accession numberWP_039428968), Bacteroides pyogenes Cas13b (accession numberWP_034542281), and Riemerella anatipestifer Cas13b (accession numberWP_004919755).
 60. The composition of claim 59, wherein the Type VI-BCas protein is Porphyromonas gulae Cas13b (accession numberWP_039434803), or Prevotella sp. P5-125 Cas13b (accession numberWP_044065294).
 61. The composition of claim 51, wherein the Type VI-BCas protein contains one or more mutations within a HEPN domaincorresponding to R116, H121, R1177, or H1182 corresponding to the aminoacid positions of Type VI-B Bergeyella zoohelcum ATCC
 43767. 62. Thecomposition of claim 51, wherein the one or more polynucleotidemolecules comprise one or more regulatory elements operably configuredto express the polypeptides and/or the nucleic acid component(s),optionally wherein the one or more regulatory elements comprise apromoter(s) or inducible promotor(s).
 63. The composition of claim 62,wherein the one or more polynucleotide molecules are comprised withinone or more vectors.
 64. The composition of claim 62, wherein thecomposition is comprised in a delivery system.
 65. A method of targetinga locus of interest in a eukaryotic cell, the method comprisingdelivering to said locus a non-naturally occurring or engineeredcomposition non-naturally occurring or engineered composition comprisingi) a Type VI-B Cas protein optimized for activity in a mammalian cell;and ii) one or more nucleic acid components, the one or more nucleicacid components capable of forming a complex with the Type VI-B Casprotein and comprising a guide sequence capable of directing sitespecific binding of the complex to a target RNA polynucleotide sequence.66. The method of claim 65, wherein the targeting is in vivo or ex vivo.67. The method of claim 65, wherein the Type VI-B Cas protein is fusedto one or more localization signals.
 68. The method of claim 67, whereinthe localization signal is a nuclear localization signal (NLS) or anuclear export signal (NES)
 69. The method of claim 65, wherein the TypeVI-B Cas protein contains one or more mutations within a HEPN domaincorresponding to R116, H121, R1177, or H1182 corresponding to the aminoacid positions of Type VI-B Bergeyella zoohelcum ATCC
 43767. 70. Themethod of claim 65, wherein the Type VI-B Cas protein is associated withone or more functional domains.
 71. The method of claim 71, wherein thefunctional domain is cytosine or adenosine deaminase functional domainassociated with the Type VI-B Cas protein, and the target RNA sequencecomprises one or more Adenine or Cytidine.
 72. A detection compositioncomprising: a non-target polynucleotide comprising a detectable marker;a Type VI Cas protein or one or more nucleic acid sequences encoding theType VI Cas protein; and at least one guide polynucleotide designed toform a complex with a CRISPR-Cas protein and comprising a guide sequencecapable of hybridizing with a target sequence, or one or more nucleicsequences encoding the at least one guide polynucleotide.
 73. A methodof detecting target nucleic acid in a sample, comprising: contacting oneor more samples comprising one or more target sequences with; i) a TypeVI Cas protein; ii) at least one guide polynucleotide designed to form acomplex with the Type VI Cas protein and comprising a guide sequence,wherein the guide sequence is capable of hybridizing with a targetsequence; and iii) a non-target polynucleotide comprising a detectablemarker; and detecting a signal from cleavage of the non-target sequence,thereby detecting the one or more target nucleic acid sequences in thesample.